Cytokine receptors associated with myelogenous haematological proliferative disorders and uses thereof

ABSTRACT

The disclosure relates to methods and compositions effective in the diagnosis, prognosis and treatment of human hematopoietic cancers. In particular, the disclosure provides tumor-associated genes that encode for cytokine receptors that are differentially expressed in hematopoietic tumor cells of myeloid origin compared with other cells, e.g., normal stem cells.

This application is the U.S. National Stage entry under §371 ofInternational Application No. PCT/US2009/038459, filed Mar. 26, 2009,which claims the benefit of priority under 35 U.S.C. §119 of U.S.application Ser. No. 61/039,701 (filed Mar. 26, 2008), each of which isincorporated herein by reference in its entirety.

1. TECHNICAL FIELD

The present disclosure relates generally to agents capable ofspecifically targeting cancer stem cell markers and methods of using theagents, particularly in diagnostic and therapeutic treatments. Inparticular, the present disclosure provides cytokine receptors as novelcancer stem cell targets that are expressed extracellularly and that aretargeted by antibodies and other agents disclosed herein.

2. BACKGROUND

The cells of the hematopoietic system arise from multipotentprogenitors, the hematopoietic stem cells (HSCs), which progress througha series of developmental programs to ultimately form the terminallydifferentiated cells of the myeloid or lymphoid lineage. It is believedthat in the initial stages of hematopoiesis, HSCs commit to twodistinguishable oligopotent but developmentally restricted progenitorcell types, the common lymphoid progenitors (CLPs) and the commonmyeloid progenitor (CMPs). T lymphocytes, B lymphocytes, natural killer(NK) cells, and lymphoid dendritic cells develop from correspondingprogenitor cells derived from the CLPs whereas erythroid cells,megakaryocytes, granulocytes, macrophages, and myeloid dendritic cellsdevelop from their corresponding progenitor cells derived from CMPs.Cell populations at each stage of differentiation are distinguishablefrom other cell populations in the hematopoietic pathway based onprogrammed expression of a unique set of cell markers.

Although HSCs are capable of self renewal—cell division that results inat least one of the daughter cells having the same characteristics asthe parent cell—the progenitor cells committed to the lymphoid ormyeloid lineages lose their potential to self-renew. That is, mitoticcell division of the committed progenitors leads to differentiatedprogeny rather than generation of a cell with the same proliferative anddifferentiation capacity as the parent cell. This loss of self-renewalpotential is seen in the ability of committed progenitors cells tomaintain hematopoiesis only for a limited time period (i.e., short termreconstitution) following transplantation of the progenitor cells intoan immunocompromised animal, as compared to an HSC, which can completelyregenerate and maintain hematopoiesis during the life of the host animal(i.e., long term reconstitution).

It has been observed, however, that in certain disease states of thehematopoietic system, dysregulation of cellular regulatory pathways maylead to progenitor cells that acquire the ability to self-renew. Forinstance, acute myeloid leukemia (AML, also called acute myelogenousleukemia) is a myeloproliferative disorder marked, in part, byinfiltration of bone marrow by abnormal hematopoietic cells. Indeed, thestem cell nature of cancer was first shown in AML (Lapidot et al., 1994Nature 17:645-8). AML is categorized into different subtypes based onmorphological features and cytochemical staining properties, andalthough the self-renewal characteristic in most types of AML isattributable to leukemic cells having cell marker phenotypes consistentwith HSCs (Bonnet, D. and Dick, J. E., Nat. Med. 3(7):730-737 (1997)),the chromosomal abnormality associated with the AML M3 subtype isobserved in cell populations with a cell marker phenotype characteristicof more differentiated cells of the myeloid lineage (CD34⁻, CD38⁺)whereas the HSC population in M3 does not carry the translocation(Turhan, A. G. et al., Blood 79:2154-2161 (1995)).

Gain of self-renewing characteristic in the committed progenitor cellpopulation is also suggested in chronic myeloid leukemia (CML, alsocalled chronic myelogenous leukemia, or chronic granulocytic leukemia),a disease commonly associated with the Philadelphia chromosome, which isa balanced translocation between chromosomes 9 and 22, t(9;22). Thetranslocation produces a fusion between the bcr and c-abl genes andresults in expression of a chimeric protein BCR-ABL with increasedtyrosine kinase activity. Although the HSC population in CML typicallycontains the chromosomal abnormality, the BCR-ABL fusion protein ismainly expressed in the committed cells of myelomonocytic lineage ratherthan the HSCs, indicating that committed cells in the myeloid lineagemay be the source of the leukemic cells rather than the HSCs. Additionalevidence for the committed myeloid cells as being the source of theleukemic clones in CML comes from studies of controlled expression ofBCR-ABL in transgenic animals. Use of promoters active specifically inmyeloid progenitor cells to force expression of BCR-ABL in committedcells but not in HSCs produces disease characteristic of CML in thesetransgenic animal models (Jaiswal. S. et al., Proc. Natl. Acad. Sci. USA100:10002-10007 (2003)).

Although myeloproliferative disorders, such as AML and CML are typicallyassociated with cytogenetic abnormalities, the cytogenetic defect maynot be solely responsible for the proliferative trait. In someinstances, the chromosomal abnormality is observed in normal cells,which suggests that accumulation of additional mutations in either theHSCs or committed myeloid cells is required for full manifestation ofthe disease state. Even in CML, the disorder displays a multiphasiccourse, beginning from a chronic phase, which after 3-5 years and up to10 years, leads to an accelerated or blastic phase similar to AML. Thetime period required to transition from the chronic phase (less than 5%blasts or promyelocytes) toa the blastic phase (>30% blasts in theperipheral blood or bone marrow) may reflect the time needed toaccumulate the mutations responsible for conversion of the chronic phaseto the more aggressive blastic phase. For the most part, however, theleukemic cells appear to retain the cell marker phenotypes detectable innormal progenitor cells.

Treatments for proliferative disorders normally rely on the sensitivityof proliferating cells to cytotoxic or cytostatic chemotherapeuticagents. For instance, busulfan, a bifunctional alkylating agent, andhydroxyurea, an inhibitor of ribonucleoside diphosphate, affect DNAsynthesis and stability, resulting in toxicity to dividing cells. Othertherapeutic agents of similar activity include cytosine arabinoside(cytarabine) and daunorubicin. However, the effects of these agents arenon-discriminatory and as a result they have serious side effects due totoxicity to normal dividing cells.

Another treatment used in patients with haematological malignancies isbone marrow transplant (BMT), where the recipient's hematopoietic cellsare eliminated with radiation and/or chemotherapy (e.g.,cyclophosphamide), and the hematopoietic system reconstituted bytransplant of healthy hematopoietic stem cells. Typically, thetransplant uses HLA matched allogeneic bone marrow cells from a familymember (HLA-identical) or a serologically matched altruistic donor(MUD). Approximately, <50% of recipients find a donor, with exactlymatching histocompatibility. Transplant with less well matched donorsmarketed increases the transplant related morbidity and mortality. Thistherapeutic approach has limited application because of its dependenceon the availability of suitable donors and because the treatments showbetter outcome for patients in the chronic or early phase of the diseaseas compared to acute or late stages.

Antibody therapy for cancer involves the use of antibodies, or antibodyfragments, against an antigen to target antigen-expressing tumor cells.Because antibody therapy targets cells expressing a particular antigen,there is a possibility of cross-reactivity with normal cells and canlead to detrimental results. Substantial efforts have been directed tofinding tumor-specific antigens. Tumor-specific antigens are foundalmost exclusively on tumors or are expressed at a greater level intumor cells than the corresponding normal cells. Thus, tumor-specificantigens provide targets for antibody targeting of cancer, or otherdisease-related, cells expressing the antigen, as well as providingmarkers for diagnosis, for example, by identifying increased levels ofexpression. In immunotherapy approaches, antibodies specific to suchtumor-specific antigens can be conjugated to cytotoxic compounds or canbe used alone in immunotherapy.

Immunotherapy as a treatment option against hematpoietic cancers, suchas AML, is limited by the lack of tumor-associated antigens that aretumor-specific and that are shared among diverse patients. It isdesirable to find other therapeutic agents that take advantage of thedevelopmental origins of the leukemic cells by exploiting the commoncharacteristics between leukemic cells and normal cell populations inthe myeloid lineage. This approach would provide treatments that cansupplement traditional therapies for myeloid leukemias, or that can beused as an alternative treatment to directly target the stem cellfractions of leukemic cells. This approach also provides additionaldiagnostic and prognostic strategies, as well as strategies formonitoring the efficacy of a therapeutic regimen.

Generally, therapeutic treatment is more effective when tailored to aspecific type of hematopoietic cancer. Predicting and determiningefficacy of a treatment regime over time is also valuable in terms ofclinical management. It is thus desirable to find tumor-specific markersthat can be used in more efficient and accurate diagnosis and prognosisof myeloiod leukemic disorders, such as AML.

Cytokine receptors belong to families of receptor proteins, which aredivided into two subsets on the basis of the presence or absence ofparticular sequence motifs. The two subsets are thehematopoietin-receptor family (also referred to as the class I cytokinereceptor family) and the class II cytokine receptor superfamily (many ofwhich are receptors for interferons or interferon-like cytokines). Inthe hematopoietin-receptor family, the α chain often defines ligandspecificity of the receptor and the β or γ chain initiates intracellularsignaling.

3. SUMMARY

The present invention provides methods and compositions effective in thediagnosis and treatment of human hematopoietic cancers of myeloidorigin. As described herein, the following cytokine receptor markershave been found to be associated with hematopoietic tumor cells (HTCs)of myeloid origin: colony stimulating factor 1 receptor (CFS1R);interleukin 13 receptor, alpha 1 (IL13RA1); interleukin 1 receptoraccessory protein (IL1RAP); interferon-α receptor 1 (IFNAR1);interleukin-5 receptor alpha (IL5RA); insulin receptor (INSR);interleukin 1 receptor-like 1 (IL1RL1); leukocyte receptor tyrosine(LTK); and tumor associated calcium signal transducer 1 (TACSTD1).

In preferred embodiments, the disclosed cytokine receptor markers aredifferentially expressed in HTCs of myeloid origin compared to normalHSCs. In some embodiments, the disclosed cytokine receptor markers aredifferentially expressed by at least about 2 fold. In other embodiments,the cytokine receptor markers are differentially expressed by at leastabout 3 fold. In other embodiments, the cytokine receptor markers aredifferentially expressed by at least about 5 fold, etc. The presentdisclosure provides agents specifically directed to these markers thatfind use in therapeutic and diagnostic applications.

The following cytokine receptor markers are over-expressed on thesurface of myelogenous HTCs: colony stimulating factor 1 receptor(CFS1R); interleukin 13 receptor, alpha 1 (IL13RA1); interleukin 1receptor accessory protein (IL1RAP); interferon-α receptor 1 (IFNAR1);interleukin-5 receptor alpha (IL5RA); insulin receptor (INSR);interleukin 1 receptor-like 1 (IL1RL1); leukocyte receptor tyrosine(LTK); and tumor associated calcium signal transducer 1 (TACSTD1).Agents specifically directed to these markers can specifically bind HTCsof myeloid origin by virtue of binding to the surface-expressed marker.

Compositions that specifically target the disclosed myelogenous HTCmarkers (e.g., the cytokine receptors disclosed herein), interferingwith the expression thereof or binding to the expressed products, areprovided herein, as well as methods of using the same in the diagnosis,prognosis and treatment of haematological proliferative disorderscharacterized by such markers. The compositions include antibodies thatspecifically bind one or more of the extracellularly-expressed antigensassociated with myelogenous HTCs that can inhibit their proliferationand/or mediate their destruction. The invention further providesimmortal cell lines that produce one or more such antibodies.

In one aspect, the invention provides antibodies that specifically bindto one or more of colony stimulating factor 1 receptor (CFS1R);interleukin 13 receptor, alpha 1 (IL13RA1); interleukin 1 receptoraccessory protein (IL1RAP); interferon-α receptor 1 (IFNAR1);interleukin-5 receptor alpha (IL5RA); insulin receptor (INSR);interleukin 1 receptor-like 1 (IL1RL1); leukocyte receptor tyrosine(LTK); and tumor associated calcium signal transducer 1 (TACSTD1)associated with HTCs of myeloid origin. In some embodiments, theantibody is a monoclonal antibody, for example, an antibody which isproduced from a hybridoma cell line. In preferred embodiments, themonoclonal antibody specifically binds to hematopoietic tumor cells ofmyeloid origin including, without limitation, chronic myeloid leukemia(CML) blasts, acute myeloid leukemia (AML) blasts, as well as to cellsfrom the KG-1a, Pfeiffer, MOLT-3, GA-10, Ramos, and Jurkat cell lines.In another embodiment, the monoclonal antibody specifically binds to AMLblasts.

In preferred embodiments, the invention provides antibodies thatspecifically bind to one or more of the disclosed cytokine receptorsassociated with myelogenous HTCs and thereby inhibit their proliferationand/or mediate their destruction, but do not mediate destruction ofnormal hematopoietic stems cells. In a preferred embodiment, theantibody is a monoclonal antibody. In some embodiments, the antibody isan IgG isotype or a humanized antibody. In one embodiment, the humanizedantibody is from a transgenic animal that includes a humanimmunoglobulin gene.

In another embodiment, the invention provides an antibody complex havingat least one antibody that specifically binds to one or more of thedisclosed cytokine receptors associated with HTCs of myeloid origin. Ina preferred embodiment, the antibody complex comprises a multimercomprising a monoclonal antibody that binds to one of the disclosedcytokine receptors

In alternative embodiments, the antibodies of the present inventioninclude detectable moieties, radioactive compounds (e.g. radioisotopesor radionuclides), or bioactive compounds (e.g. drugs or smallmolecules). In some embodiments, the bioactive compound is a cytotoxicagent.

In another embodiment, the invention provides small molecules, whichbind, preferably specifically, to one or more of the cytokine receptorpolypeptides disclosed herein. In preferred embodiments, the smallmolecule is a small organic molecule, including small organic moleculesknown in the art as being an agonist or antagonist of a polypeptidecorresponding to a cytokine receptor disclosed herein. Small moleculesknown to bind polypeptides corresponding to other cytokine receptorsdisclosed herein can also find use in the subject therapeutic,prognostic and/or diagnostic applications.

Optionally, the small molecule is conjugated to a growth inhibitoryagent or cytotoxic agent such as a toxin, including, for example, amaytansinoid or calicheamicin, an antibiotic, a radioactive isotope, anucleolytic enzyme, or the like. The small molecules that find use inthe therapeutic methods of the instant invention preferably induce deathof a cell to which they bind. For diagnostic purposes, the smallmolecules can be detectably labeled and/or attached to a solid support.

The subject agents and antibodies directed to the disclosed cytokinereceptors have significant therapeutic and diagnostic utilities and inadditional aspects pharmaceutical compositions, methods and kits areprovided employing the subject agents and antibodies for use indiagnosing and treating haematological proliferative disorderscharacterized by the presence of one or more of the disclosed cytokinereceptors such as, e.g., acute myelogenous leukemia, acutemyelomonocytic leukemia, chronic myelogenous leukemia and acute myeloidleukemia.

In one aspect, the present disclosure provides methods of usingantibodies to target one or more of the disclosed cytokine receptors. Inthe present teachings, the antibodies provide a basis forimmunotherapeutic approaches in treating disorders involvinghematopoietic tumor cells (HTCs) of myeloid origin, for example,myeloproliferative disorders such as chronic myeloid leukemia (CML) andacute myeloid leukemia (AML).

In one embodiment, the present invention provides methods of inhibitingthe proliferation of HTCs of myeloid orign by contacting the HTCs with acomposition comprising an antibody or other agent directed to one ormore of the disclosed cytokine receptors. In another embodiment, thepresent invention provides methods of mediating the destruction of HTCsof myeloid origin by contacting the HTCs with a composition comprisingan antibody or other agent directed to one or more of the disclosedcytokine receptors. In one embodiment, the antibody is a monoclonalantibody that specifically binds an epitope on a disclosed cytokinereceptor or a portion thereof. In another embodiment, the compositioncomprises an antibody complex.

In another embodiment, a method of depleting HTCs over-expressing one ormore of the disclosed cytokine receptors in a subject in need thereof isprovided in which the subject is administered a composition comprisingan antibody or antibody complex as described herein. In yet anotherembodiment, the present invention provides a method of treating apatient with a myelogenous haematological proliferative disordercharacterized by over-expression in HTCs of one or more of the disclosedcytokine receptors where the patient is administered a composition thatincludes an antibody or antibody complex or other agent as describedherein.

In some embodiments, the methods of the present invention are suitablefor treating a haematological proliferative disorder of myeloid orgin,including myoproliferative disorders such as, for example, chronicmyeloid leukemia (CML) and/or acute myeloid leukemia (AML).

In another aspect, the present invention provides diagnostic methods forhematological proliferative disorders of myeloid origin, where the levelof an expression product corresponding to one or more of the disclosedcytokine receptors is detected. In one embodiment, the expressionproduct is a transcription product, such as RNA. Methods of detectingthe level of RNA include utilizing a specific hybridization probe or anarray of such probes. In another embodiment, the expression product is atranslation product such as one or more of the cytokine receptorantigens corresponding to one or more of the cytokine receptorsdisclosed herein. Methods of detecting the level of an antigen includeutilizing antibodies of the instant disclosure. The RNA or antigen levelcan be compared to control levels, e.g., levels obtained from samples ofnormal HSCs.

In another embodiment, the present invention provides prognostic methodsfor predicting the efficacy of treating a haematological proliferativedisorder of myeloid origin, where the level of an expression productcorresponding to one or more of the disclosed cytokine receptors isdetected and wherein the expression product level is correlated with atreatment outcome. In one embodiment, the expression product is RNA andlower expression levels correlate with more favorable outcomes.

In still another embodiment the present invention provides methods formonitoring the efficacy of treating a haematological proliferativedisorder of myeloid origin, where the level of an expression productcorresponding to one or more of the disclosed cytokine receptors isdetected at various time points; and where a change in the level iscorrelated with treatment outcome. In one embodiment, the expressionproduct is RNA and decreasing levels indicate a positive response totreatment.

In some embodiments, the methods of the present invention are suitablefor diagnosis, prognosis and monitoring of a haematologicalproliferative disorder of myeloid origin, such as myoproliferativedisorders. The present invention provides methods of diagnosis,prognosis and monitoring of chronic myeloid leukemia (CML) and/or acutemyeloid leukemia (AML). In a particularly preferred embodiment, thehaematological proliferative disorder is AML and the level of RNA orantigen is detected using a test sample comprising AML HTCs, which iscompared to control levels obtained from a control sample of normalHSCs.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of double sorting Lin⁻CD34⁺CD90⁺CD45RA⁻CD38⁻and Lin⁻CD34⁺CD90⁺CD45RA⁻CD38⁺ cells from 3 samples taken from 3individual mobilized peripheral blood (MPB) donors that are notafflicted with AML.

FIG. 2 shows the results of of double sorting Lin-CD34+CD90+CD45RA−CD38−and Lin−CD34+CD90+CD45RA−CD38+ cells from 3 samples taken fromperipheral blood of 3 individual patients diagnosed with AML.

FIG. 3 shows representative results of incubating peripheral blood cellsfrom an individual diagnosed with AML with an antibody specific forIL1RAP. The upper left panels show the gating, and bottom right panelsshow IL1RAP staining.

FIG. 4 shows representative results of incubating CD34+ immobilizedperipheral blood from donors not afflicted with AML.with an antibodyspecific for IL1RAP. The upper left panels show the gating, and bottomand right panels show IL1RAP staining.

FIG. 5 shows representative results of incubating peripheral blood cellsfrom an individual diagnosed with AML with an antibody specific forIL13R1. The upper left panels show the gating, and bottom and rightpanels show IL13R1 staining.

FIG. 6 shows representative results of incubating CD34+ immobilizedperipheral blood from donors not afflicted with AML.with an antibodyspecific for IL13R1. The upper left panels show the gating, and bottomand right panels show IL13R1 staining.

5. DETAILED DESCRIPTION OF EMBODIMENTS 5.1 Definitions

For the following descriptions, the technical and scientific terms usedherein will have the meanings commonly understood by one of ordinaryskill in the art, unless specifically defined otherwise. Accordingly,the following terms are intended to have the following meanings:

The terms “cancer stem cell (CSC) markers” or “cancer stem cell (CSC)targets” as well as “hematopoietic tumor cell (HTC) markers” or“hematopoietic tumor cell (HTC) targets” refer to genes and theirexpression products, such as mRNA and polypeptides, that have been foundto be associated with HTCs by virture, for example, of increasedexpression and/or biological activity. For example, in the case of theCFS1R marker disclosed herein, CFS1R mRNA transcribed from the CFS1Rgene is found at higher levels in samples comprising AML HTCs ascompared with samples comprising normal HSCs. An HTC associated with agiven marker is referred to herein as a “marker+ HTC.”

“HTCs of myeloid origin” particularly refers to cancer stem cellsderived from cells of the myeloid (nonlymphoid) lineages, includingmonocytes, macrophages, neutrophils, basophils, eosinophils,erythrocytes, megakaryocytes/platelets, dendritic cells and the like.HTCs of myeloid origin will be those found in myeloid leukemias, such asAML and CML, where it is believed that progenitor cells commited tomyeloid lineages regain self-renewing characteristics. Various forms ofleukemia, for example, appear to have their origins in a smallpopulation of HSCs or committed myeloid progenitor cells in which thecells acquire a combination of mutations that give rise to the malignantphenotype. The terms “HTCs of myeloid orign” and “myeloid HTCs” or“myelogenous HTCs” are used herein interchangeably.

“Hematopoietic stem cell” or “HSC” generally refers to clonogenic, selfrenewing pluripotent cells, capable of ultimately differentiating intoall cell types of the hematopoietic system, including B cells T cells,NK cells, lymphoid dendritic cells, myeloid dendritic cells,granulocytes, macrophages, megakaryocytes, and erythroid cells. As withother cells of the hematopoietic system, HSCs are typically defined bythe presence of a characteristic set of cell markers.

“Marker phenotyping” refers to identification of markers or antigens oncells for determining its phenotype (e.g., differentiation state and/orcell type). This may be done by immunophenotyping, which uses antibodiesthat recognize antigens present on a cell. The antibodies may bemonoclonal or polyclonal, but are generally chosen to have minimalcrossreactivity with other cell markers. It is to be understood thatcertain cell differentiation or cell surface markers are unique to theanimal species from which the cells are derived, while other cellmarkers will be common between species. These markers definingequivalent cell types between species are given the same markeridentification even though there are species differences in structure(e.g., amino acid sequence). Cell markers include cell surfacesmolecules, also referred to in certain situations as celldifferentiation (CD) markers, and gene expression markers. The geneexpression markers are those sets of expressed genes indicative of thecell type or differentiation state. In part, the gene expression profilewill reflect the cell surface markers, although they may includenon-cell surface molecules.

Lineage markers are cell surface antigens that can be used forimmunophenotyping cells of a particular developmental lineage. Forexample, a set of ‘Lin’ antigens comprising CD2, CD3, CD4, CD5, CD8,NK1.1, B220, TER-119, Gr-1 can be used to identify mature murine bloodcells. Cells that do not express these marker antigens, or express themat very low levels, are said to be lineage marker negative (Lin⁻). Themonoclonal antibody cocktails directed against these lineage markers canbe used to remove cells expressing these antigens from source tissues(for example, bone marrow, umbilical cord blood, mobilized peripheralblood, fetal liver, and the like). This negative selection procedureyields a population of cells that is enriched for primitivehematopoietic stem cells or very early progenitor cells or precursorcells that do (not yet) express these markers (see, for example: KTLScells). These cells are called lineage negative cells, abbreviated Lin⁻cells. Several subpopulations of lineage negative cells have beenidentified that are enriched for hematopoietic stem cells. They includeLin⁻CD34⁺ cells (Krause et al, 1994), Lin⁻Sca⁻¹⁺c-Kit⁺Thy1^(low) cells(Fleming et al, 1993) and human CD34⁺CD38⁻ cell populations.

“Agent” refers to any molecule specifically directed to one or more ofthe disclosed HTC markers and that can act to inhibit, hinder and/orsuppress a biological activity of the HTC marker in a haematologicalproliferative disorder and/to mediate destruction of the HTCs. Agentsinclude any molecule that specifically interacts with an HTC marker geneand/or expression product, including for example, antibodies thatspecifically bind to an antigen corresponding to a HTC marker to inhibitHTC proliferation and/or mediate their destruction; antisense moleculesthat interfere with the expression of an HTC marker; or molecules thatinterfere with a biological activity mediated by the HTC marker, such asby sterically inhibiting interaction between an HTC marker and itsligand to interfere with activation of a cancer stem cell signaltransduction pathway. The molecule may be one known in the art, e.g.,small molecule agonists or antagonists directed towards one or more ofthe HTC markers disclosed herein. An antibody that specifcally binds toan antigen corresponding to an HTC marker disclosed herein is referredto as a “marker specific antibody.”

A “small molecule”, “small molecule compound”, or “small organicmolecule” refers to a molecule having a molecular weight usually lessthan about 2000 daltons, alternatively less than about 1500, about 750,about 500, about 250 or about 200 daltons in size, wherein suchmolecules are known in the art to be capable of binding (preferablyspecifically binding) to a polypeptide corresponding to an cytokinereceptor disclosed herein.

With regard to the binding of a small molecule compound to a targetmolecule, the term “specifically interacts with” or “specifically binds”or is “directed to or towards” a particular product means binding thatis measurably different from a non-specific interaction. Specificbinding can be measured, for example, by methods known in the art, e.g.,using competition assays with a control molecule that is similar to thetarget, for example, an excess of non-labeled target. A small moleculecompound that specifically binds a target can have a Kd for the targetof at least about 10⁻⁴ M, alternatively at least about 10⁻⁵ M,alternatively at least about 10⁻⁶ M, alternatively at least about 10⁻⁷M, alternatively at least about 10⁻⁸ M, alternatively at least about10⁻⁹ M, alternatively at least about 10⁻¹⁰ M, alternatively at leastabout 10⁻¹¹ M, alternatively at least about 10⁻¹² M, or greater. In oneembodiment, the term “specific binding” refers to binding where a smallmolecule compound binds to its particular target without substantiallybinding to any other polypeptide or macromolecule.

“Antibody” refers to a composition comprising a protein that bindsspecifically to a corresponding antigen and has a common, generalstructure of immunoglobulins. The term antibody specifically coverspolyclonal antibodies, monoclonal antibodies, dimers, multimers,multispecific antibodies (e.g., bispecific antibodies), and antibodyfragments, so long as they exhibit the desired biological activity.Antibodies may be murine, human, humanized, chimeric, or derived fromother species. Typically, an antibody will comprise at least two heavychains and two light chains interconnected by disulfide bonds, whichwhen combined form a binding domain that interacts with an antigen. Eachheavy chain is comprised of a heavy chain variable region (V_(H)) and aheavy chain constant region (C_(H)). The heavy chain constant region iscomprised of three domains, C_(H1), C_(H2) and C_(H3), and may be of themu, delta, gamma, alpha or epsilon isotype. Similarly, the light chainis comprised of a light chain variable region (V_(L)) and a light chainconstant region (C_(L)). The light chain constant region is comprised ofone domain, C_(L), which may be of the kappa or lambda isotype. TheV_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The constant regions of the antibodies may mediate the bindingof the immunoglobulin to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system. The heavy chainconstant region mediates binding of the immunoglobulin to host tissue orhost factors, particularly through cellular receptors such as the Fcreceptors (e.g., FcγRI, FcγRII, FcγRIII, etc.). As used herein, antibodyalso includes an antigen binding portion of an immunoglobulin thatretains the ability to bind antigen. These include, as examples, F(ab),a monovalent fragment of V_(L) C_(L) and V_(H) C_(H) antibody domains;and F(ab)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region. The term antibody alsorefers to recombinant single chain Fv fragments (scFv) and bispecificmolecules such as, e.g., diabodies, triabodies, and tetrabodies (see,e.g., U.S. Pat. No. 5,844,094).

Antibodies may be produced and used in many forms, including antibodycomplexes. As used herein, the term “antibody complex” or “antibodycomplexes” is used to mean a complex of one or more antibodies withanother antibody or with an antibody fragment or fragments, or a complexof two or more antibody fragments. Antibody complexes include multimericforms of antibodies directed to the disclosed cytokine receptors such ashomoconjugates and heteroconjugates as well as other cross-linkedantibodies as describd herein.

“Antigen” is to be construed broadly and refers to any molecule,composition, or particle that can bind specifically to an antibody. Anantigen has one or more epitopes that interact with the antibody,although it does not necessarily induce production of that antibody.

The terms “cross-linked”, “cross-linking” and grammatical equivalentsthereof, refer to the attachment of two or more antibodies to formantibody complexes, and may also be referred to as multimerization.Cross-linking or multimerization includes the attachment of two or moreof the same antibodies (e.g. homodimerization), as well as theattachment of two or more different antibodies (e.g.heterodimerization). Those of skill in the art will also recognize thatcross-linking or multimerization is also referred to as forming antibodyhomoconjugates and antibody heteroconjugates. Such conjugates mayinvolve the attachment of two or more monoclonal antibodies of the sameclonal origin (homoconjugates) or the attachment of two or moreantibodies of different clonal origin (also referred to asheteroconjugates or bispecific). Antibodies may be crosslinked bynon-covalent or covalent attachment. Numerous techniques suitable forcross-linking will be appreciated by those of skill in the art.Non-covalent attachment may be achieved through the use of a secondaryantibody that is specific to the primary antibody species. For example,a goat anti-mouse (GAM) secondary antibody may be used to cross-link amouse monoclonal antibody. Covalent attachment may be achieved throughthe use of chemical cross-linkers.

“Epitope” refers to a determinant capable of specific binding to anantibody. Epitopes are chemical features generally present on surfacesof molecules and accessible to interaction with an antibody. Typicalchemical features are amino acids and sugar moieties, havingthree-dimensional structural characteristics as well as chemicalproperties including charge, hydrophilicity, and lipophilicity.Conformational epitopes are distinguished from non-conformationalepitopes by loss of reactivity with an antibody following a change inthe spatial elements of the molecule without any change in theunderlying chemical structure.

“Humanized antibody” refers to an immunoglobulin molecule containing aminimal sequence derived from non-human immunoglobulin. Humanizedantibodies include human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, a humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the CDR regions correspond to those of anon-human immunoglobulin and all or substantially all of the framework(FR) regions are those of a human immunoglobulin consensus sequence. Ahumanized antibody will also encompass immunoglobulins comprising atleast a portion of an immunoglobulin constant region (Fc), generallythat of a human immunoglobulin (Jones et al., Nature 321:522-525 (1986);Reichmann et al, Nature 332:323-329 (1988)).

“Immunogen” refers to a substance, compound, or composition whichstimulates the production of an immune response.

The term “immunoglobulin locus” refers to a genetic element or set oflinked genetic elements that comprise information that can be used by aB cell or B cell precursor to express an immunoglobulin peptide. Thispeptide can be a heavy chain peptide, a light chain peptide, or thefusion of a heavy and a light chain peptide. In the case of anunrearranged locus, the genetic elements are assembled by a B cellprecursor to form the gene encoding an immunoglobulin peptide. In thecase of a rearranged locus, a gene encoding an immunoglobulin peptide iscontained within the locus.

“Isotype” refers to an antibody class defined by its heavy chainconstant region. Heavy chains are generally classified as gamma, mu,alpha, delta, epsilon and designated as IgG, IgM, IgA, IgD, and IgE.Variations within each isotype are categorized into subtypes, forexample subtypes of IgG are divided into IgG₁, IgG₂, IgG₃, and IgG₄,while IgA is divided into IgA₁ and IgA₂. The IgY isotype is specific tobirds.

“Monoclonal antibody” or “monoclonal antibody composition” refers to apreparation of antibody molecules of single molecular composition. Amonoclonal antibody composition displays a single binding specificityand affinity for a particular epitope.

The term “human monoclonal antibody” refers to antibodies displaying asingle binding specificity which have variable and/or constant regions(if present) derived from human germline immunoglobulin sequences. Inone embodiment, the human monoclonal antibodies are produced by ahybridoma which includes a B cell obtained from a transgenic non-humananimal, e.g., a transgenic mouse, having a genome comprising a humanheavy chain transgene and a light chain transgene fused to animmortalized cell.

“Single chain Fv” or “scFv” refers to an antibody comprising the V_(H)and V_(L) regions of an antibody, wherein these domains are present in asingle polypeptide chain. Generally, a scFv further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thescFv to form the desired structure for antigen binding.

“Specifically immunoreactive” or antibody that “specifically binds”refers to a binding reaction of the antibody that is determinative ofthe presence of the antigen in a heterogeneous population of antigens.The antibody may be described as being “directed to” or “directedagainst” the particular antigen. Under a designated immunoassaycondition, the antibody binds to the antigen at least two times, andtypically 10-1000 times or more over background. “Specificallyimmunoreactive” or “antibody that specifically binds” also refers to anantibody that is capable of binding to an antigen with sufficientaffinity such that the antibody is useful in targeting a cell having theantigen bound to its surface or in targeting the soluble antigen itself.In such embodiments, the extent of non-specific binding is the amount ofbinding at or below background and will typically be less than about10%, preferably less than about 5%, and more preferably less than about1% as determined by fluorescence activated cell sorting (FACS) analysisor radioimmunoprecipitation (RIA), for example.

“Recombinant antibody” refers to all antibodies prepared and expressed,created or isolated by recombinant techniques. These include antibodiesobtained from an animal that is transgenic for the immunoglobulin locus,antibodies expressed from a recombinant expression vector, or antibodiescreated, prepared, and expressed by splicing of any immunoglobulin genesequence to other nucleic acid sequences.

The term “associated with” as in, for example, cytokine receptor markersbeing “associated with” hematopoietic tumor cells (HTCs), refers to thecase where the HTC marker genes (e.g., the cytokine receptor genes) areexpressed at differential levels in HTCs as opposed to other mammaliancells, e.g., normal HSCs. That is, a transcription product, such as RNA,and/or a translation product, such as a polypeptide, corresponding tothe cytokine receptor gene has been found at differential levels in oneor more samples comprising HTCs compared with one or more samplescomprising other mammalian cells, e.g., normal HSCs.

“Expression” or “expressing” as used herein refers to bothtranscriptional and translational processes directed by a gene. That is,the terms refer to the process of converting genetic information encodedin a nucleic acid sequence (gene) into RNA (e.g, mRNA rRNA, tRNA, snRNA,etc) through transcription of the gene; and/or convereting geneticinformation into protein through translation of mRNA. Similarly,“expression product” as used herein refers to a trancription ortranslation product of a gene, and includes, e.g., RNA (mRNA, tRNA,rRNA, snRNA, etc.,) as well as polypeptides (intracellular,extracellular or surface expressed proteins).

“Differential expression”, “differentially expressed”, “differentiallevels”, and the like, as used herein refers to a difference in thelevel of an expression product corresponding to a marker in HTCs incomparison to other mammalian cells, e.g., normal HSCs in particular.The difference can be expressed as an expression ratio or signal ratio,obtained from the quotient of the level of expression of an expressionproduct in HTCs over the level of expression of the same expressionproduct in HSCs. “Differential expression” generally refers to adifference in expression levels of at least about 2 fold, at least about3 fold, at least about 5 fold, at least about 7 fold, at least about 10fold, or at least about 15 fold. In particularly preferred embodiments,the difference in expression levels is at least about 20 fold, at leastabout 30 fold, at least about 40 fold, or at least about 50 fold. Instill more preferred embodiments, the difference in expression levels isat least about 70 fold, at least about 100 fold, at least about 200fold, or as much as nearly 300 fold, 400 fold or 500 fold.

The term “extracellularly-expressed” refers to the case where expressionproducts are found outside of a cell, whether existing entirely outsideof the plasma membrane of a cell (as in the case of secreted, solubleproducts); or existing partly outside of a cell as in the case of somemembrane- (or surface-) expressed products. Membrane-bound proteins maybe integral or peripheral, so long as at least a portion of the proteinis accessible to antibodies outside the cell. The term membrane-boundincludes membrane-expressed products as well as receptor bound productsthat become associated with surface membranes by virtue of binding to amembrane-bound receptor.

“Subject” or “patient” are used interchangeably and refer to, exceptwhere indicated, mammals such as humans and non-human primates, as wellas rabbits, rats, mice, goats, pigs, and other mammalian species.

“Haematological proliferative disorder” or “hematopoietic proliferativedisorder” refers to a condition characterized by the clonalproliferation of one or more hematopoietic cells of the myeloid and/orlymphoid lineage. “Hematological proliferative disorer of the myeloidlineage” refers to conditions characterized by the clonal proliferationprimarily of one or more hematopoietic cells of the myeloid lineage,rather than the lymphoid lineage. It is to be noted that herein the term“of myeloid origin” is used interchangeably with the adjectives“myeloid” or “myelogenous.” Myelogenous hematopoietic proliferativedisorders include, e.g., the general classes of (a) dysmyelopoieticdisease, (b) acute myeloproliferative leukemia and (c) chronicmyeloproliferative disease. Each general class is further categorizedinto different subtypes, as is known in the art.

5.2 Hybridomas and Monoclonal Antibodies

The teachings of the present disclosure provide hybridoma cell lines andmonoclonal antibodies that specifically bind to one or more of thefollowing cytokine receptor products: colony stimulating factor 1receptor (CFS1R); interleukin 13 receptor, alpha 1 (IL13RA1);interleukin 1 receptor accessory protein (IL1RAP); interferon-α receptor1 (IFNAR1); interleukin-5 receptor alpha (IL5RA); insulin receptor(INSR); interleukin 1 receptor-like 1 (IL1RL1);leukocyte receptortyrosine (LTK); and tumor associated calcium signal transducer 1(TACSTD1) associated with hematopoietic tumor cells (HTCs) of myeloidorigin.

The following cytokine receptor markers are over-expressed on thesurface of HTCs of myeloid origin: colony stimulating factor 1 receptor(CFS1R); interleukin 13 receptor, alpha 1 (IL13RA1); interleukin 1receptor accessory protein (IL1RAP); interferon-α receptor 1 (IFNAR1);interleukin-5 receptor alpha (IL5RA); insulin receptor (INSR);interleukin 1 receptor-like 1 (IL1RL1); leukocyte receptor tyrosine(LTK); and tumor associated calcium signal transducer 1 (TACSTD1). Theinvention provides anti-CFS1R antibodies; anti-IL13RA1 antibodies;anti-IL1RAP antibodies; anti-IFNAR1 antibodies; anti-IL5RA antibodies;anti-INSR antibodies; anti-IL1RL1 antibodies; anti-LTK antibodies; andanti-TACSTD1 antibodies, where the antibodies are preferably monoclonalantibodies.

CFS1R is a disulfide-linked homodimer, also referred to in the art asCD115, 1436, CFS1R, 164770, P07333, C-FMS, CSFR, FIM2 and FMS, andformerly referred to as McDonough feline sarcoma viral (v-fms) oncogenehomolog. CFS1R is normally expressed on myeloid cells, in particularprogenitors and monocytes, and osteoclasts as a membrane boundpolypeptide. Upon binding to its ligand CSF-1, the receptor undergoestyrosine autophosphorylation and subsequently, phosphorylates othermembrane-proximal downstream targets, resulting in a number ofphysiological effects, including cytoskeletal remodeling, genetranscription and protein translation. CFS1R activation is known topromote the survival, proliferation and differentiation of mononuclearphagocytes, as well as the spreading and motility of macrophages. Inosteoclasts, CFS1R synergizes with RANKL to regulate the differentiationof mononuclear phagocytes to osteoclasts.

The present invention provides anti-CFS1R antibodies, preferablymonoclonal antibodies, that can specifically bind to CFS1R antigen,e.g., CFS1R polypeptide exposed on the surface of HTCs of myeloidorigin. As discussed in more detail below, the anti-CFS1R antibodiespreferably bind such myelogenous HTCs, thereby inhibiting theirproliferation and/or mediating their destruction. One of skill in theart will further recognize that antibodies known in the art can find usein the methods disclosed in the instant invention. For example, one ormore anti-CFS1R antibodies commercially available from ABCAM®, ABGENT®,ABNOVA®, ANTIGENIX AMAERICA®, EBIOSCIENCE®, GENETEX®, LAB VISION®,LIFESPAN BIOSCIENCES®, MILLIPORE®, NOVUS® BIOLOGICALS, R&D SYSTEMS®,SIGMA-ALDRICH® AND THERMO SCINETIFIC PIERCE®may also find use withrespect to diagnostic and/or therapeutic applications taught herein.

IL13RA1 occurs as 4 different splice variants and is normally found on Tand B cells, as well as on endothelial cells. It is also referred to asCD213a1, CD213A1, NR4, IL13RA, IL-13R, IL-13RA and IL-13R-alpha-1. TheIL13RA1 polypeptide is a type I transmembrane protein, which interactswith IL4R to form IL13 receptor. The IL13 receptor in turn binds IL13,and can partially replace the common gamma chain in an IL-2 recptorcomplex.

The present invention provides anti-IL13RA antibodies, preferablymonoclonal antibodies, that can specifically bind to IL13RA antigen,e.g., IL13RA polypeptide exposed on the surface of HTCs of myeloidorigin. As discussed in more detail below, the anti-IL13RA antibodiespreferably bind such myelogenous HTCs, thereby inhibiting theirproliferation and/or mediating their destruction. One of skill in theart will further recognize that antibodies known in the art can find usein the methods disclosed in the instant invention. For example, one ormore anti-IL13RA antibodies commercially available from ABCAM®, ABNOVA®,ABR-AFFINITY BIOREAGENTS®, ATLAS ANTIBODIES®, CELL SCIENCES®, DIACLONE®,GENETEX®, LIFESPAN BIOSCIENCES®, NOVUS® BIOLOGICALS, R&D SYSTEMS®, andSTRATEGIC DIGNOSTICS®may also find use with respect to diagnostic and/ortherapeutic applications taught herein.

IL1 RAP, also known as IL1 R3, IL-1 RAcP, FLJ37788, occurs as 11alternative splice forms, and is normally expressed onmacrophages/monocytes, B cells, platelets, thymocytes, T cells anddendritic cells. IL1RAP is a type I transmembrane protein, but alsooccurs as a secreted version. The protein is known to generally mediateIL-1 dependent activation of NF-κB.

The present invention provides anti-IL1RAP antibodies, preferablymonoclonal antibodies, that can specifically bind to IL1RAP antigen,e.g., IL1RAP polypeptide exposed on the surface of HTCs of myeloidorigin. As discussed in more detail below, the anti-IL1RAP anitbodiespreferably bind such myelogenous HTCs, thereby inhibiting theirproliferation and/or mediating their destruction. One of skill in theart will further recognize that antibodies known in the art can find usein the methods disclosed in the instant invention. For example, one ormore anti-IL1RAP antibodies commercially available from ABCAM®,ABNOVA®CORPORATION, GENETEX®, LIFESPAN BIOSCIENCES®, ANDNOVUS®BIOLOGICALS may also find use with respect to diagnostic and/ortherapeutic applications taught herein.

IFNAR1 is a type I membrane protein that forms one of the two chains ofa receptor for interferons alpha and beta. Binding and activation of thereceptor stimulates Janus protein kinases, which in turn phosphorylateseveral proteins, including STAT1 and STAT2. It may also function as anantiviral factor. IFNAR1 is also known as AVP, IFRC, IFNBR,IFN-alpha-REC, IFNAR.

The present invention provides anti-IFNAR1 antibodies, preferablymonoclonal antibodies, that can specifically bind to IFNAR1 antigen,e.g., IFNAR1 polypeptide exposed on the surface of HTCs of myeloidorigin. As discussed in more detail below, the anti-IFNAR1antibodiespreferably bind such myelogenous HTCs, thereby inhibiting theirproliferation and/or mediating their destruction. One of skill in theart will further recognize that antibodies known in the art can find usein the methods disclosed in the instant invention. For example, one ormore anti-IFNAR1 antibodies commercially available from ABCAM®,NOVUS®BIOLOGICALS, and R&D SYSTEMS® may also find use with respect todiagnostic and/or therapeutic applications taught herein.

IL5RA is an interleukin 5 specific subunit of a heterodimeric cytokinereceptor. The IL5 heterodimeric receptor comprises a ligand specificalpha subunit and a signal transducing beta subunit shared by thereceptors for interleukin 3 (IL3) and colony stimulating factor 2(CSF2/GMCSF). The beta subunit is activated by ligand binding, and isrequired for biological activity of IL5. IL5RA may interact with thesyndecan binding protein (syntenin), which is required for IL5 mediatedactivation of the transcription factor SOX4. Six alternatively splicedvariants encoding three distinct isoforms have been reported. IL5RA isalso known as CD125, CDw125, HSIL5R3, and MGC26560.

The present invention provides anti-IL5RA antibodies, preferablymonoclonal antibodies, that can specifically bind to IL5RA antigen,e.g., IL5RA polypeptide exposed on the surface of HTCs of myeloidorigin. As discussed in more detail below, the anti-IL5RA antibodiespreferably bind such myelogenous HTCs, thereby inhibiting theirproliferation and/or mediating their destruction. One of skill in theart will further recognize that antibodies known in the art can find usein the methods disclosed in the instant invention. For example, one ormore anti-IL5RA antibodies commercially available from ABCAM®, THERMOSCIENTIFIC PIERCE®, GENETEX®, PROTEINTECH®, and ATLAS ANTIBODIES®mayalso find use with respect to diagnostic and/or therapeutic applicationstaught herein.

INSR, also known as HHF5 and CD220, stimulates glucose uptake.

The present invention provides anti-INSR antibodies, preferablymonoclonal antibodies, that can specifically bind to INSR antigen, e.g.,INSR polypeptide exposed on the surface of HTCs of myeloid origin. Asdiscussed in more detail below, the anti-INSR antibodies preferably bindsuch myelogenous HTCs, thereby inhibiting their proliferation and/ormediating their destruction. One of skill in the art will furtherrecognize that antibodies known in the art can find use in the methodsdisclosed in the instant invention. For example, one or more anti-INSRantibodies commercially available from R&D DIAGNOSTICS® and LIFESPAN®may also find use with respect to diagnostic and/or therapeuticapplications taught herein.

IL1RL1, also known as T1, ST2, DER4, ST2L, ST2V, FIT-1, and MGC32623, isa member of the interleukin 1 receptor family. It may be induced byproinflammatory stimuli, and may be involved in the function of helper Tcells.

The present invention provides anti-IL1RL1 antibodies, preferablymonoclonal antibodies, that can specifically bind to IL1RL1 antigen,e.g., IL1RL1 polypeptide exposed on the surface of HTCs of myeloidorigin. As discussed in more detail below, the anti-IL1RL1 antibodiespreferably bind such myelogenous HTCs, thereby inhibiting theirproliferation and/or mediating their destruction. One of skill in theart will further recognize that antibodies known in the art can find usein the methods disclosed in the instant invention. For example, one ormore anti-IL1RL1 antibodies commercially available from PROTEIN TECH®may also find use with respect to diagnostic and/or therapeuticapplications taught herein.

LTK is a member of the ros/insulin receptor family of tyrosine kinases.Multiple transcript variants encoding different isoforms have been foundfor this gene. LTK is also known as TYK1.

The present invention provides anti-LTK antibodies, preferablymonoclonal antibodies, that can specifically bind to LTK antigen, e.g.,LTK polypeptide exposed on the surface of HTCs of myeloid origin. Asdiscussed in more detail below, the anti-LTK antibodies preferably bindsuch myelogenous HTCs, thereby inhibiting their proliferation and/ormediating their destruction. One of skill in the art will furtherrecognize that antibodies known in the art can find use in the methodsdisclosed in the instant invention. For example, one or more anti-LTKantibodies commercially available from ABCAM®, ABGENT,®, ABNOVA®,LIFESPAN BIOSCIENCES®, NOVUS® BIOLOGICALS, and SANTA CRUZBIOLOGICALS®may also find use with respect to diagnostic and/ortherapeutic applications taught herein.

TACSTD1 is expressed on most normal epithelial cells andgastrointestinal carcinomas. It may function as a homotypiccalcium-independent cell adhesion molecule and is used as a target forimmunotherapy of human carcinomas. TACSTD1 is also known as EGP, ESA,KSA, M4S1, MK1, EGP2, EGP34, EGP40, KS1/4, MIC18, TROP1, CO-17A, EpCAM,hEGP2, CO17-1A, GA733-2, and TACST-1.

The present invention provides anti-TACSTD1 antibodies, preferablymonoclonal antibodies, that can specifically bind to TACSTD1 antigen,e.g., TACSTD1 polypeptide exposed on the surface of HTCs of myeloidorigin. As discussed in more detail below, the anti-TACSTD1 antibodiespreferably bind such myelogenous HTCs, thereby inhibiting theirproliferation and/or mediating their destruction. One of skill in theart will further recognize that antibodies known in the art can find usein the methods disclosed in the instant invention. For example, one ormore anti-TACSTD1 antibodies commercially available from LIFESPANBIOSCIENCES® may also find use with respect to diagnostic and/ortherapeutic applications taught herein.

Monoclonal antibodies of the instant disclosure specifically bindmyelogenous HTCs by virtue of specifc binding to its target antigen. Inpreferred embodiments, the monoclonal antibody (or a derivative thereof)is specifically immunoreactive with cells of myeloid origin thehematopoietic system, such as granulocyte/macrophage progenitors (GMP),KG-1a, K-562, Jurkat, CML blasts, and AML blasts. For example, in somesuch embodiments, specific binding to HTCs, by virtue of a cytokinereceptor disclosed herein, mediates destruction of hematopoietic tumorcells of myeloid origin.

Antibodies can be produced readily by one skilled in the art. Thegeneral methodology for making monoclonal antibodies by hybridomas isnow well known to the art. See, e.g., M. Schreier et al., HybridomaTechniques (Cold Spring Harbor Laboratory); Hammerling et al.,Monoclonal Antibodies and T-Cell Hybridomas (Elsevier Biomedical Press).As described above, the present disclosure provides methods of producingthe monoclonal antibodies or derivatives thereof. In some embodiments,these methods comprise cultivating a hybridoma cell under suitableconditions, wherein the antibody is produced and obtaining the antibodyand/or derivative thereof from the cell and/or from the cell culturemedium. A specific example of making the monoclonal antibodies of theinstant invention is also provided in the Examples below.

The antibodies can be purified by methods known to the skilled artisan.Purification methods include, among others, selective precipitation,liquid chromatography, HPLC, electrophoresis, chromatofocusing, andvarious affinity techniques. Selective precipitation may use ammoniumsulfate, ethanol (Cohn precipitation), polyethylene glycol, or othersavailable in the art. Liquid chromatography mediums, include, amongothers, ion exchange medium DEAE, polyaspartate), hydroxylapatite, sizeexclusion (e.g., those based on crosslinked agarose, acrylamide,dextran, etc.), hydrophobic matrixes (e.g., Blue Sepharose). Affinitytechniques typically rely on proteins that interact with theimmunoglobulin Fc domain. Protein A from Staphylococcus aureas can beused to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein Gfrom C and G streptococci is useful for all mouse isotypes and for humanγ3 (Guss et al., EMBO J. 5:15671575 (1986)). Protein L, aPeptostreptococcus magnus cell-wall protein that binds immunoglobulins(Ig) through k light-chain interactions (BD Bioscience/ClonTech. PaloAlto, Calif.), is useful for affinity purification of Ig subclasses IgM,IgA, IgD, IgG, IgE and IgY. Recombinant forms of these proteins are alsocommercially available. If the antibody contains metal binding residues,such as phage display antibodies constructed to contain histidine tags,metal affinity chromatography may be used. When sufficient amounts ofspecific cell populations are available, antigen affinity matrices maybe made with the cells to provide an affinity method for purifying theantibodies.

The present invention provides the antibodies described herein, as wellas corresponding antibody fragments and antigen-binding portions. Theterms “antibody fragment” or “antigen-binding portion” of an antibody(or simply “antibody portion”) of the present invention, as used herein,refers to one or more fragments of an antibody that retain the abilityto specifically bind to an antigen. It has been shown that theantigen-binding function of an antibody can be performed by fragments ofa full-length antibody. Examples of binding fragments encompassed withinthe term “antibody fragment” or “antigen-binding portion” of an antibodyinclude (i) a Fab fragment, a monovalent fragment consisting of theV_(L), V_(H), C_(L) and C_(H1) domains; (ii) a F(ab′)₂ fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; (iii) a Fd fragment consisting of the V_(H)and C_(H1) domains; (iv) a Fv fragment consisting of the V_(L) and V_(H)domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,(1989) Nature 341:544-546), which consists of a V_(H) domain; and (vi)an isolated complementarity determining region (CDR), and (vii)bispecific single chain Fv dimers (PCT/US92/09965). Furthermore,although the two domains of the Fv fragment, V_(L) and V_(H), are codedfor by separate genes, they can be joined, using recombinant methods, bya synthetic linker that enables them to be made as a single proteinchain in which the V_(L) and V_(H) regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are screened forutility in the same manner as are intact antibodies. The antibodyfragments may be modified. For example, the molecules may be stabilizedby the incorporation of disulphide bridges linking the VH and VL domains(Reiter et al., 1996, Nature Biotech. 14:1239-1245).

The present disclosure further provides fragments of the antibodiesdisclosed herein. Immunoglobulin molecules can be cleaved intofragments. The antigen binding region of the molecule can be dividedinto either F(ab′)₂ or Fab fragments. The F(ab′)₂ fragment is divalentand is useful when the Fc region is either undesirable or not a requiredfeature. The Fab fragment is univalent and is useful when an antibodyhas a very high avidity for its antigen. Eliminating the Fc region fromthe antibody decreases non-specific binding between the Fc region and Fcreceptor bearing cells. To generate Fab or F(ab)₂ fragments, theantibodies are digested with an enzyme. Proteases that cleave at thehinge region of an immunoglobulin molecule preserve the disulfidebond(s) linking the F(ab) domain such that they remain togetherfollowing cleavage. A suitable protease for this purpose is pepsin. Forproducing F(ab) fragments, proteases are chosen such that cleavageoccurs above the hinge region containing the disulfide bonds that jointhe heavy chains but which leaves intact the disulfide bond linking theheavy and light chain. A suitable protease for making F(ab) fragments ispapain. The fragments are purified by the methods described above, withthe exception of affinity techniques requiring the intact Fc region(e.g., Protein A affinity chromatography).

Antibody fragments can be produced by limited proteolysis of antibodiesand are called proteolytic antibody fragments. These include, but arenot limited to, the following: F(ab′)₂ fragments, Fab′ fragments,Fab′-SH fragments, and Fab fragments. “F(ab′)₂ fragments” are releasedfrom an antibody by limited exposure of the antibody to a proteolyticenzyme, e.g., pepsin or ficin. A F(ab′)₂fragment comprises two “arms,”each of which comprises a variable region that is directed to andspecifically binds a common antigen. The two Fab′ molecules are joinedby interchain disulfide bonds in the hinge regions of the heavy chains;the Fab′ molecules may be directed toward the same (bivalent) ordifferent (bispecific) epitopes. “Fab′ fragments” contain a singleanti-binding domain comprising a Fab and an additional portion of theheavy chain through the hinge region. “Fab′-SH fragments” are typicallyproduced from F(ab′)₂ fragments, which are held together by disulfidebond(s) between the H chains in an F(ab′)₂ fragment. Treatment with amild reducing agent such as, by way of non-limiting example,beta-mercaptoethylamine, breaks the disulfide bond(s), and two Fab′fragments are released from one F(ab′)₂ fragment. Fab′-SH fragments aremonovalent and monospecific. “Fab fragments” (i.e., an antibody fragmentthat contains the antigen-binding domain and comprises a light chain andpart of a heavy chain bridged by a disulfide bond) are produced bypapain digestion of intact antibodies. A convenient method is to usepapain immobilized on a resin so that the enzyme can be easily removedand the digestion terminated. Fab fragments do not have the disulfidebond(s) between the H chains present in a F(ab′)₂ fragment.

“Single-chain antibodies” are one type of antibody fragment. The termsingle chain antibody is often abbreviated as “scFv” or “sFv.” Theseantibody fragments are produced using molecular genetics and recombinantDNA technology. A single-chain antibody consists of a polypeptide chainthat comprises both a V_(H) and a V_(L) domains which interact to forman antigen-binding site. The V_(H) and V_(L) domains are usually linkedby a peptide of 10 to 25 amino acid residues.

The term “single-chain antibody” further includes but is not limited toa disulfide-linked Fv (dsFv) in which two single-chain antibodies (eachof which may be directed to a different epitope) linked together by adisulfide bond; a bispecific sFv in which two discrete scFvs ofdifferent specificity is connected with a peptide linker; a diabody (adimerized sFv formed when the V_(H) domain of a first sFv assembles withthe V_(L) domain of a second sFv and the V_(L) domain of the first sFvassembles with the V_(H) domain of the second sFv; the twoantigen-binding regions of the diabody may be directed towards the sameor different epitopes); and a triabody (a trimerized sFv, formed in amanner similar to a diabody, but in which three antigen-binding domainsare created in a single complex; the three antigen binding domains maybe directed towards the same or different epitopes).

“Complementary determining region peptides” or “CDR peptides” areanother form of an antibody fragment. A CDR peptide (also known as“minimal recognition unit”) is a peptide corresponding to a singlecomplementarity-determining region (CDR), and can be prepared byconstructing genes encoding the CDR of an antibody of interest. Suchgenes are prepared, for example, by using the polymerase chain reactionto synthesize the variable region from RNA of antibody-producing cells.See, for example, Larrick et al., Methods: A Companion to Methods inEnzymology 2:106, 1991.

In “cysteine-modified antibodies,” a cysteine amino acid is inserted orsubstituted on the surface of antibody by genetic manipulation and usedto conjugate the antibody to another molecule via, e.g., a disulfidebridge. Cysteine substitutions or insertions for antibodies have beendescribed (see U.S. Pat. No. 5,219,996). Methods for introducing Cysresidues into the constant region of the IgG antibodies for use insite-specific conjugation of antibodies are described by Stimmel et al.(J. Biol. Chem 275:330445-30450, 2000).

The present disclosure further provides humanized and non-humanizedantibodies. Humanized forms of non-human (e.g., mouse) antibodies arechimeric antibodies that contain minimal sequence derived from non-humanimmunoglobulin. Generally, humanized antibodies are non-human antibodiesthat have had the variable-domain framework regions swapped forsequences found in human antibodies. The humanized antibodies may behuman immunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Generally, in a humanized antibody, the entire antibody, except theCDRs, is encoded by a polynucleotide of human origin or is identical tosuch an antibody except within its CDRs. The CDRs, some or all of whichare encoded by nucleic acids originating in a non-human organism, aregrafted into the beta-sheet framework of a human antibody variableregion to create an antibody, the specificity of which is determined bythe engrafted CDRs. The creation of such antibodies is described in,e.g., WO 92/11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al.,1988, Science 239:1534-1536. Humanized antibodies can also be generatedusing mice with a genetically engineered immune system. Roque et al.,2004, Biotechnol. Prog. 20:639-654.

The present disclosure further provides humanized and non-humanizedantibodies. Humanized forms of non-human (e.g., mouse) antibodies arechimeric antibodies that contain minimal sequence derived from non-humanimmunoglobulin. Generally, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

It can be desirable to modify the antibodies of the invention withrespect to effector function, so as to enhance, e.g., the effectivenessof the antibody in treating cancer. For example, cysteine residue(u)canbe introduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedcan have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176:1191-1195 (1992)and Shopes, J. Immunol., 148:2918-2922 (1992). Homodimeric antibodieswith enhanced anti-tumor activity can also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53:2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and can thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3:219-230 (1989).

In preferred embodiments, the antibodies described herein specificallybind to an antigen corresponding to one or more of the disclosedcytokine receptors present on the cell surface of hematopoietic tumorcells (HTCs) that arose from progenitor cell populations in the myeloidlineage of the hematopoietic system. Differentiation in the myeloidlineage leads to formation of terminally differentiated cells thatinclude, among others, megakaryocytes, erythroid cells, macrophages,basophils, eosinophils, neutrophils and myeloid dendritic cells. Thesecells originate from hematopoietic stem cells (HSC), which differentiatethrough a series of progenitor cell populations displaying progressivelyrestricted differentiation potential. The HSCs and the progenitor cellpopulations are identifiable from each other based on, among otherdistinguishing characteristics, their capacity to differentiate intospecific cell subsets and the presence of a particular set of cellularmarkers that is specific to the cell population. In some embodiments,the monoclonal antibodies of the present disclosure are directed toprogenitor cells of the myeloid lineage that are marker+ HTCs. In someembodiments, the monoclonal antibodies in the present disclosure aredirected to committed myeloid progenitor cells that are marker+ HTCs

5.2.1 Modified Antibodies

In another aspect, the present invention provides modified antibodiesthat are derived from an antibody that specifically binds an antigencorresponding to a cytokine receptor disclosed herein. Modifiedantibodies also include recombinant antibodies as described herein.

Numerous types of modified or recombinant antibodies will be appreciatedby those of skill in the art. Suitable types of modified or recombinantantibodies include without limitation, engineered murine monoclonalantibodies (e.g. murine monoclonal antibodies, chimeric monoclonalantibodies, humanized monoclonal antibodies), domain antibodies (e.g.Fab, Fv, V_(H), scFV, and dsFv fragments), multivalent or multispecificantibodies (e.g. diabodies, minibodies, miniantibodies, (scFV)₂,tribodies, and tetrabodies), and antibody conjugates as describedherein.

In one aspect, the present invention includes domain antibodies. “Domainantibodies” are functional binding domains of antibodies, correspondingto the variable regions of either the heavy (VH) or light (VL) chains ofhuman antibodies. Domain antibodies may have a molecular weight ofapproximately 13 kDa, or less than one-tenth the size of a fullantibody. They are well expressed in a variety of hosts includingbacterial, yeast, and mammalian cell systems. In addition, domainantibodies are highly stable and retain activity even after beingsubjected to harsh conditions, such as freeze-drying or heatdenaturation. See, for example, U.S. Pat. No. 6,291,158; 6,582,915;6,593,081; 6,172,197; US Serial No. 2004/0110941; European Patent0368684; U.S. Pat. No. 6,696,245, WO04/058821, WO04/003019 andWO03/002609. In one embodiment, the domain antibody of the presentinvention is a single domain. Single domain antibodies may be prepared,for example, as described in U.S. Pat. No. 6,248,516, incorporatedherein by reference in its entirety. In some embodiments, the presentinvention provides domain antibodies derived from an antibody thatspecifically binds to an antigen corresponding to one of the cytokinereceptors disclosed herein.

In another aspect, the present invention includes multi-specificantibodies. Multi-specific antibodies include bispecific, trispecific,etc. antibodies. Bispecific antibodies can be produced via recombinantmeans, for example by using leucine zipper moieties (i.e., from the Fosand Jun proteins, which preferentially form heterodimers; Kostelny etal., 1992, J. Immnol. 148:1547) or other lock and key interactive domainstructures as described in U.S. Pat. No. 5,582,996. Additional usefultechniques include those described in U.S. Pat. Nos. 5,959,083; and5,807,706. In one embodiment, the present invention providesmulti-specific antibodies that include an antibody that specificallybinds an antigen corresponding to a cytokine receptor disclosed herein.In another embodiment, the multispecific antibody is bispecific.

Bispecific antibodies are also sometimes referred to as “diabodies.”These are antibodies that bind to two (or more) different antigens. Alsoknown in the art are triabodies (a trimerized sFv, formed in a mannersimilar to a diabody, but in which three antigen-binding domains arecreated in a single complex; the three antigen binding domains may bedirected towards the same or different epitopes) or a tetrabodies (fourantigen-binding domains created in a single complex where the fourantigen binding domains may be directed towards the same or differentepitopes), and the like. Dia-, tria- and tetrabodies can be manufacturedin a variety of ways known in the art (Holliger and Winter, 1993,Current Opinion Biotechnol. 4:446-449), e.g., prepared chemically orfrom hybrid hybridomas. In addition, such antibodies and fragmentsthereof may be constructed by gene fusion (Tomlinson et. al., 2000,Methods Enzymol. 326:461-479; WO94/13804; Holliger et al., 1993, Proc.Natl. Acad. Sci. U.S.A. 90:6444-6448, each of which is incorporatedherein by reference in their entirety). In one embodiment, the diabody,triabody, or tetrabody is derived from an antibody that specificallybinds an antigen corresponding to a cytokine receptor disclosed herein.In a preferred embodiment, the diabody, triabody, or tetrabody isderived from two or more monoclonal antibodies that specifically bind todifferent antigens, each antigen corresponding to different cytokinereceptors disclosed herein.

In another embodiment, the present invention provides minibodies, whichare minimized antibody-like proteins that include a scFV joined to a CH3domain, that are derived from an antibody that specifically binds anantigen corresponding to a cytokine receptor disclosed herein.Minibodies can be made as described in the art (Hu et al., 1996, CancerRes. 56:3055-3061).

In another embodiment, the present invention provides bindingdomain-immunglobulin fusion proteins, where the binding domainspecifically binds an antigen corresponding to a HTC marker disclosedherein. The fusion protein may include a marker specific binding domainpolypeptide fused to an immunoglobulin hinge region polypeptide, whichis fused to an immunoglobulin heavy chain CH2 constant regionpolypeptide fused to an immunoglobulin heavy chain CH3 constant regionpolypeptide. Under the present invention, marker specific antibodyfusion proteins can be made by methods appreciated by those of skill inthe art (See published U.S. Patent Application Nos. 20050238646,20050202534, 20050202028, 2005020023, 2005020212, 200501866216,20050180970, and 20050175614, each of which is incorporated herein byreference in their entirety).

In another embodiment, the present invention provides a heavy-chainprotein (V_(HH)) derived from a marker specific antibody.Naturally-occuring heavy chain antibodies (e.g. camelidae antibodieshaving no light chains) have been utlitized to develop antibody-derivedtherapeutic proteins that typically retain the structure and functionalproperties of naturally-occuring heavy-chain antibodies. They are knownin the art as Nanobodies®. Under the present invention, heavy chainproteins (V_(HH)) derived from a marker specific heavy chain antibodymay be made by methods appreciated by those of skill in the art (Seepublished U.S. Patent Application Nos. 20060246477, 20060211088,20060149041, 20060115470, and 20050214857, each of which is incorporatedherein by reference in their entirety).

In one aspect, the present invention provides a modified antibody thatis a human antibody. In one embodiment, the marker specific antibodiesdescribed herein are fully human antibodies. “Fully human antibody ” or“complete human antibody” refers to a human antibody having only thegene sequence of an antibody derived from a human chromosome. Theanti-human marker specific complete human antibody can be obtained by amethod using a human antibody-producing mouse having a human chromosomefragment containing the genes for a heavy chain and light chain of ahuman antibody [see Tomizuka, K. et al., Nature Genetics, 16, p.133-143, 1997; Kuroiwa, Y. et al., Nuc. Acids Res., 26, p. 3447-3448,1998; Yoshida, H. et al., Animal Cell Technology: Basic and AppliedAspects vol. 10, p.69-73 (Kitagawa, Y., Matuda, T. and Iijima, S. eds.),Kluwer Academic Publishers, 1999; Tomizuka, K. et al., Proc. Natl. Acad.Sci. USA, 97, 722-727, 2000] or obtained by a method for obtaining ahuman antibody derived from a phage display selected from a humanantibody library (see Wormstone, I. M. et al., InvestigativeOphthalmology & Visual Science. 43(7), p.2301-8, 2002; Carmen, S. etal., Briefings in Functional Genomics and Proteomics, 1 (2), p.189-203,2002; Siriwardena, D. et al., Ophthalmology, 109(3), p.427-431, 2002).

In one aspect, the present invention provides a marker specific antibodythat is an antibody analog, sometimes referred to as “syntheticantibodies.” For example, a variety of recent work utilizes eitheralternative protein scaffolds or artificial scaffolds with grafted CDRs.Such scaffolds include, but are not limited to, mutations introduced tostabilize the three-dimensional structure of the binding protein as wellas wholly synthetic scaffolds consisting for example of biocompatiblepolymers. See, for example, Korndorfer et al., 2003, Proteins:Structure, Function, and Bioinformatics, Volume 53, Issue 1:121-129.Roque et al., 2004, Biotechnol. Prog. 20:639-654. In addition, peptideantibody mimetics (“PAMs”) can be used, as well as work based onantibody mimetics utilizing fibronection components as a scaffold.

5.2.2 Cross-linked Antibodies

In one aspect, the present invention provides cross-linked antibodiesthat include two or more antibodies described herein attached to eachother to form antibody complexes. Cross-linked antibodies are alsoreferred to as antibody multimers, homoconjugates, and heteroconjugates.It has been observed in the art that the multimerization of an antibodypreviously observed to have no signalling activity can result in amultimerized antibody with potent signalling activity. This has beenparticularly noted in the field of anti-tumor agents. For example, ithas been reported that the IgG-IgG homodimerization of anti-CD19,anti-CD20, anti-CD21, anti-CD22, and anti-Her-2 monoclonal antibodiesconfers potent anti-tumor ability to such homodimers (Ghetie, M. et al.(1997) Proc. Natl. Acad. Sci., USA, Vol. 94, pp-7509-7514 incorporatedherein by reference in its entirety). In addition, the homodimerizationof monoclonal antibodies known to have anti-tumor activity, such asRituximab®, can lead to an increase in effectiveness as an anti-tumoragent (Ghetie, M. (2001) Blood, Vo. 97;5: 1392-1398 incorporated hereinby reference in its entirety).

In some embodiments, the antibody complexes provided herein includemultimeric forms of antibodies directed to one or more of the antigenscorresponding to one or more of the cytokine receptors disclosed herein.For example, antibodies complexes of the invention may take the form ofantibody dimers, trimers, or higher-order multimers of monomericimmunoglobulin molecules. Crosslinking of antibodies can be done throughvarious methods know in the art. For example, crosslinking of antibodiesmay be accomplished through natural aggregation of antibodies, throughchemical or recombinant linking techniques or other methods known in theart. For example, purified antibody preparations can spontaneously formprotein aggregates containing antibody homodimers, and otherhigher-order antibody multimers. In one embodiment, the presentinvention provides homodimerized antibodies that specifically bind to anantigen corresponding to a cytokine receptor disclosed herein.

Antibodies can be cross-linked or dimerized through linkage techniquesknown in the art (see Ghetie et al. (1997) supra ; Ghetie et al. (2001)supra). Non-covalent methods of attachment may be utilized. In aspecific embodiment, crosslinking of antibodies can be achieved throughthe use of a secondary crosslinker antibody. The crosslinker antibodycan be derived from a different animal compared to the antibody ofinterest. For example, a goat anti-mouse antibody (Fab specific) may beadded to a mouse monoclonal antibody to form a heterodimer. Thisbivalent crosslinker antibody recognizes the Fab or Fc region of the twoantibodies of interest forming a homodimer.

In one embodiment of the present invention, an antibody thatspecifically binds to an antigen corresponding to a cytokine receptordisclosed herein is cross-linked using a goat anti-mouse antibody (GAM).In another embodiment, the GAM crosslinker recognizes the Fab or Fcregion of two antibodies, each of which specifically binds the same ortwo different antigens corresponding to the same or different cytokinereceptors disclosed herein.

Methods for covalent or chemical attachment of antibodies may also beutilized. Chemical crosslinkers can be homo or heterobifunctional andwill covalently bind with two antibodies forming a homodimer.Cross-linking agents are well known in the art; for example, homo-orhetero-bifunctional linkers as are well known (see the 2006 PierceChemical Company Crosslinking Reagents Technical Handbook; Hermanson, G.T., Bioconjugate Techniques, Academic Press, San Diego, Calif. (1996);Aslam M. and Dent A H., Bioconjugation: protein coupling techniques forthe biomedical sciences, Houndsmills, England: Macmillan Publishers(1999); Pierce: Applications Handbook & Catalog, Perbio Science,Ermbodegem, Belgium (2003-2004); Haughland, R. P., Handbook ofFluorescent Probes and Research Chemicals Eugene, 9^(th) Ed., MolecularProbes, OR (2003); and U.S. Pat. No. 5,747,641; all referencesincorporated herein by reference) Those of skill in the art willappreciate the suitability of various functional groups on the aminoacid(s) of an antibody for modification, including cross-linking.Suitable examples of chemical crosslinkers used for antibodycrosslinking include, but not limited to, SMCC [succinimidyl4-(maleimidomethyl)cyclohexane-1-carboxylate], SATA [N-succinimidylS-acethylthio-acetate], hemi-succinate esters of N-hydroxysuccinimide;sulfo-N-hydroxy-succinimide; hydroxybenzotriazole, and p-nitrophenol;dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (ECD), and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDCI) (see,e.g., U.S. Pat. No. 4,526,714, the disclosure of which is fullyincorporated by reference herein). Other linking reagents includeglutathione, 3-(diethoxyphosphoryloxy)-1,2,3- benzotriazin-4(3H)-one(DEPBT), onium salt-based coupling reagents, polyoxyethylene-basedheterobifunctional cross-linking reagents, and other reagents (Haitao,et al., Organ Lett 1:91-94 (1999); Albericio et al., J Organic Chemistry63:9678-9683 (1998); Arpicco et al., Bioconjugate Chem. 8:327-337(1997); Frisch et al., Bioconjugate Chem. 7:180-186 (1996); Deguchi etal., Bioconjugate Chem. 10:32-37 (1998); Beyer et al., J. Med. Chem.41:2701-2708 (1998); Drouillat et al., J. Pharm. Sci. 87:25-30 (1998);Trimble et al., Bioconjugate Chem. 8:416-423 (1997)).

Exemplary protocols for the formation of antibody homodimers is given inU.S. Patent Publication 20060062786, and Ghetie et al., (1997) supra,which are hereby incorporated by reference in their entirety. In apreferred embodiment, the chemical cross-linker used is an SMCC or SATAcrosslinker.

In addition, the antibody-antibody conjugates of this invention can becovalently bound to each other by techniques known in the art such asthe use of the heterobifunctional cross-linking reagents, GMBS(maleimidobutryloxy succinimide), and SPDP (N-succinimidyl3-(2-pyridyldithio)propionate) [see, e.g., Hardy, “Purification AndCoupling Of Fluorescent Proteins For Use In Flow Cytometry”, Handbook OfExperimental Immunology, Volume 1, Immunochemistry, Weir et al. (eds.),pp. 31.4-31.12 4th Ed., (1986), and Ledbetter et al. U.S. Pat. No.6,010,902, each of which is incorporated herein by reference in theirentirety].

In addition, antibodies may be linked via a thioether cross-link asdescribed in U.S. Patent Publication 20060216284, U.S. Pat. No.6,368,596, which is incorporated herein by reference. As will beappreciated by those skilled in the art, antibodies can be crosslinkedat the Fab region. In some embodiments, it is desirable that thechemical crosslinker not interact with the antigen-binding region of theantibody as this may affect antibody function.

5.2.3 Conjugated Antibodies

The antibodies disclosed herein can be conjugated to inorganic ororganic compounds, including, by way of example and not limitation,other proteins, nucleic acids, carbohydrates, steroids, and lipids (seefor example Green, et al., Cancer Treatment Reviews, 26:269-286 (2000).The compound may be bioactive. Bioactive refers to a compound having aphysiological effect on the cell as compared to a cell not exposed tothe compound. A physiological effect is a change in a biologicalprocess, including, by way of example and not limitation, DNAreplication and repair, recombination, transcription, translation,secretion, membrane turnover, cell adhesion, signal transduction, celldeath, and the like. A bioactive compound includes pharmaceuticalcompounds.

In one aspect, the antibodies are conjugated to or modified to carry adetectable compound. Conjugating antibodies to detectable enzymes,fluorochromes, or ligands provides a signal for visualization orquantitation of the target antigen. Antibodies may be labeled withvarious enzymes to provide highly specific probes that both visualizethe target and amplify the signal by acting on a substrate to produce acolored or chemiluminescent product. Horseradish peroxidase, alkalinephosphatase, glucose oxidase, and β-galactosidase are the commonly usedenzymes for this purpose. Fluorochromes, such as fluoresceinisothiocyanate, tetramethylrhodamine isothiocyanate (TRITC),phycoerythrin, and Cy5, provide a colored reagent for visualization anddetection. Suitable fluorescent compounds are described in Haughland, R.P., Handbook of Fluorescent Probes and Research Chemicals Eugene, 9thEd., Molecular Probes, OR (2003).

In another aspect, the conjugated compounds are chelating ligands, ormacrocyclic organic chelating compounds, particularly metal chelatingcompounds used to image intracellular ion concentrations or used ascontrast agents for medical imaging purposes. Chelating ligands areligands that can bind with more than one donor atom to the same centralmetal ion. Chelators or their complexes have found applications as MRIcontrast agents, radiopharmaceutical applications, and luminescentprobes. Conjugates of chelating compounds useful for assessingintracellular ion concentrations may be voltage sensitive dyes andnon-voltage sensitive dyes. Exemplary dye molecules for measuringintracellular ion levels include, by way of example and not limitation,Quin-2; Fluo-3; Fura-Red; Calcium Green; Calcium Orange 550 580; CalciumCrimson; Rhod-2 550 575; SPQ; SPA; MQAE; Fura-2; Mag-Fura-2; Mag-Funs-5;Di-4-ANEPPS; Di-8-ANEPPS; BCECF; SNAFL-1; SBFI; and SBFI.

In another embodiment, the ligands are chelating ligands that bindparamagnetic, superparamagnetic or ferromagnetic metals. These areuseful as contrast agents for medical imaging and for delivery ofradioactive metals to selected cells. Metal chelating ligands, include,by way of example and not limitation, diethylenetriaminepenta aceticacid (DTPA); diethylenetriaminepenta acetic acid bis(methylamide);macrocyclic tetraamine1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA); andporphyrins (see, e.g., The Chemistry of Contrast Agents in MedicalMagnetic Resonance Imaging, Merbach A. E. and Toth E., Ed., WileyInterscience (2001)). Paramagnetic metal ions, which are detectable intheir chelated form by magnetic resonance imaging, include, for example,iron(III), gadolinium(III), manganese (II and III), chromium(III),copper(II), dysprosium(III), terbium(III), holmium (III), erbium (III),and europium (III). Paramagnetic metal ions particularly useful asmagnetic resonance imaging contrast agents comprise iron(III) andgadolinium(III) metal complexes. Other paramagnetic, superparamagneticor ferromagnetic are well known to those skilled in the art.

In another embodiment, the metal-chelate comprises a radioactive metal.Radioactive metals may be used for diagnosis or as therapy based ondelivery of small doses of radiation to a specific site in the body.Targeted metalloradiopharmaceuticals are constructed by attaching theradioactive metal ion to a metal chelating ligand, such as those usedfor magnetic imaging, and delivering the chelate-complex to cells. Anexemplary radioactive metal chelate complex is DTPA (see, e.g., U.S.Pat. No. 6,010,679).

In a further aspect, the conjugated compounds are peptide tags used forpurposes of detection, particularly through the use of antibodiesdirected against the peptide. Various tag polypeptides and theirrespective antibodies are well known in the art. Examples includepoly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags;the flu HA tag polypeptide and its antibody 12CA5 (Field et al., Mol.Cell. Biol. 8:2159-2165 (1988)); the c-myc tag and the 8F9, 3C7, 6E10,G4, B7 and 9E10 antibodies thereto (Evan et al., Mol. Cell. Biol.5:3610-3616 (1985)); and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody (Paborsky et al., Protein Engineering 3:547-553(1990)). Other tag polypeptides include the Flag-peptide (Hopp et al.,BioTechnology 6:1204-1210 (1988)); the KT3 epitope peptide (Martin etal., Science 255:192-194 (1992)); tubulin epitope peptide (Skinner etal., J. Biol. Chem. 266:15163-15166 (1991)); and the T7 gene 10 proteinpeptide tag (Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA87:6393-6397 (1990)).

In another embodiment, the conjugated compounds may comprise toxins thatcause cell death, or impair cell survival when introduced into a cell. Asuitable toxin is campylobacter toxin CDT (Lara-Tejero, M., Science290:354-57 (2000)). Expression of the CdtB subunit, which has homologyto nucleases, causes cell cycle arrest and ultimately cell death.Another exemplary toxin is diptheria toxin (and similar Pseudomonasexotoxin), which functions by ADP ribosylating ef-2 (elongation factor2) molecule in the cell and preventing translation. Entry of thediptheria toxin A subunit induces cell death in cells containing thetoxin fragment. Other useful toxins include cholera toxin and pertussistoxin (catalytic subunit-A ADP ribosylates the G protein regulatingadenylate cyclase), pierisin from cabbage butterflys, an inducers ofapoptosis in mammalian cells (Watanabe, M., Proc. Natl. Acad. Sci. USA96:10608-13 (1999)), ribosome inactivating toxins (e.g., ricin A chain,Gluck, A. et al., J. Mol. Biol. 226:411-24 (1992)); and nigrin (Munoz,R. et al., Cancer Lett. 167: 163-69 (2001)).

Bioactive compounds suitable for delivery by the compositions herein,include, among others, chemotherapeutic compounds, including by way ofexample and not limitation, vinblastin, bleomycin, taxol, cis-platin,adriamycin, and mitomycin. Exemplary chemotherapeutic agents suitablefor the present purposes are compounds acting on DNA synthesis andstability. For example, anti-neoplastic agents of the anthracyclin classof compounds act by causing strand breaks in the DNA and are used asstandard therapy against cancer. Exemplary anti-neoplastic agents ofthis class are daunorubicin and doxorubicin. Coupling of these compoundsto proteins, including antibodies, are described in Langer, M. et al.,J. Med. Chem. 44(9):1341-1348 (2001) and King, H. D. et al., Bioconjug.Chem. 10:279-288 (1999)). By attaching or linking the antineoplasticagents to the antibodies, the compounds are delivered to HTCs of myeloidorigin with a high degree of specificity and promote killing of thetargeted cells.

Other classes of antitumor agents are the enediyne family ofantibiotics, representative members of which include calicheamicins,neocarzinostatin, esperamincins, dynemicins, kedarcidin, andmaduropeptin (see, e.g., Smith, A. L. and Nicolaou, K. C., J. Med. Chem.39:2103-2117 (1996)). Similar to doxorubicin and daunorubicin, theantitumor activity of these agents resides in their ability to createstrand breaks in the cellular DNA. Conjugates to antibodies have beenused to deliver these molecules into those tumor cells expressingantigens recognized by the antibody and shown to have potent antitumoractivity with reduced unwanted toxicity as compared to the unconjugatedcompounds (Hinman, L. M. et al., Cancer Res. 53:3336-3342 (1993)).Conjugating the enediyne compounds to the compositions described hereinprovides another method of targeting HTCs of myeloid origin.

Radioactive compounds are useful as signals (e.g., tracers) or used toprovide a therapeutic effect by their delivery to a cell targeted (e.g.,in the form of radiopharmaceutical preparations) and may be attached tothe antibodies by methods described below. Useful radioactive nuclidesinclude, by way of example and not limitation, ³H, ¹⁴C, ³²P, ³⁵S, ⁵¹Cr,⁵⁷Co ⁵⁹Fe, ⁶⁷Ga, ⁸²Rb, ⁸⁹Sr, ⁹⁹Tc, ¹¹¹In, ¹²³I, ¹²⁵I, ¹²⁹I, ¹³¹I, and¹⁸⁶Re.

The conjugation of compounds to antibodies is well known to the skilledartisan, and typically takes advantage of functional groups present onor introduced onto the antibodies and compound. Functional groupsinclude, among others, hydroxyl, amino, thio, imino, and carboxymoieties. Reaction between functional groups may be aided by couplingreagents and crosslinking agents. Crosslinking agents and linkers andcorresponding methods for conjugation are described in Hermanson, G. T.,Bioconjugate Techniques, Academic Press, San Diego, Calif. (1996); AslamM. and Dent A H., Bioconjugation: protein coupling techniques for thebiomedical sciences, Houndsmills, England: Macmillan Publishers (1999);Pierce: Applications Handbook & Catalog, Perbio Science, Ermbodegem,Belgium (2003-2004); Haughland, R. P., Handbook of Fluorescent Probesand Research Chemicals Eugene, 9^(th) Ed., Molecular Probes, OR (2003);and U.S. Pat. No. 5,747,641; all references incorporated herein byreference. Exemplary coupling or linking reagents include, by way ofexample and not limitation, hemi-succinate esters ofN-hydroxysuccinimide; sulfo-N-hydroxy-succinimide; hydroxybenzotriazole,and p-nitrophenol; dicyclohexylcarbodiimide (DCC),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (ECD), and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDCI) (see,e.g., U.S. Pat. No. 4,526,714) the disclosure of which is fullyincorporated by reference herein. Other linking reagents includeglutathione, 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one(DEPBT), onium salt-based coupling reagents, polyoxyethylene-basedheterobifunctional cross-linking reagents, and other reagents thatfacilitate the coupling of antibodies to organic drugs and peptides andother ligands (Haitao, et al., Organ Lett 1:91-94 (1999); Albericio etal., J Organic Chemistry 63:9678-9683 (1998); Arpicco et al.,Bioconjugate Chem. 8:327-337 (1997); Frisch et al., Bioconjugate Chem.7:180-186 (1996); Deguchi et al., Bioconjugate Chem. 10:32-37 (1998);Beyer et al., J. Med. Chem. 41:2701-2708 (1998); Drouillat et al., J.Pharm. Sci. 87:25-30 (1998); Trimble et al., Bioconjugate Chem.8:416-423 (1997)).

Techniques for conjugating therapeutic compounds to antibodies are alsodescribed in Arnon et al., “Monoclonal Antibodies for Immunotargeting ofDrugs in Cancers Therapy,” in Monoclonal Antibodies and Cancer Therapy,Reisfeld et al., ed., pp 243-256, Alan R. Liss, Inc. (1985); Thorpe, etal. “The Preparation and Cytotoxic Properties of Antibody ToxinConjugates,” Immunol. Rev. 62:119-58 (1982); and Pietersz, G. A., “Thelinkage of cytotoxic drugs to monoclonal antibodies for the treatment ofcancer,” Bioconjugate Chemistry 1(2):89-95 (1990), all referencesincorporated herein by reference.

6. ANTISENCE MOLECULES AND SMALL MOLECULE COMPOUNDS

Another aspect of the present invention relates to antisense and sensemolecules comprising a singe-stranded nucleic acid sequence (either RNAor DNA) that can bind to mRNA (sense) or DNA (antisense) targetsequences corresponding to a cytokine receptor disclosed herein.Antisense or sense oligonucleotides, according to the present invention,comprise a fragment of the coding region of DNA of the genecorresponding to a cytokine receptor. Such a fragment generallycomprises at least about 14 nucleotides, preferably from about 14 toabout 30 nucleotides. Antisense or sense RNA or DNA molecules aregenerally at least about 5 nucleotides in length, alternatively at leastabout 15, at least about 30, at least about 50, at least about 100, atleast about 150, at least about 200, at least about 300, at least about400, at least about 500, at least about 700, at least about 800, or atleast about 1000 nucleotides in length. The ability to derive anantisense or a sense oligonucleotide, based upon a cDNA sequenceencoding a given protein is described in, for example, Stein and Cohen(Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechniques6:958, 1988).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block transcriptionor translation of the target sequence by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. Such methods areencompassed by the present invention. The antisense oligonucleotidesthus may be used to block the expression of proteins corresponding to acytokine receptor, wherein those marker proteins may play a role in theinduction and/or persistance of myeloid leukemias in mammals.

Preferred intragenic sites for antisense binding include the regionincorporating the translation initiation/start codon (5′-AUG/5′-ATG) ortermination/stop codon (5′-UAA, 5′-UAG and 5-UGA/5′-TAA, 5′-TAG and5′-TGA) of the open reading frame (ORF) of the gene. These regions referto a portion of the mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation initiation or termination codon. Other preferred regions forantisense binding include: introns; exons; intron-exon junctions; theopen reading frame (ORF) or “coding region,” which is the region betweenthe translation initiation codon and the translation termination codon;the 5′ cap of an mRNA which comprises an N7-methylated guanosine residuejoined to the 5′-most residue of the mRNA via a 5′-5′ triphosphatelinkage and includes 5′ cap structure itself as well as the first 50nucleotides adjacent to the cap; the 5′ untranslated region (5′UTR), theportion of an mRNA in the 5′ direction from the translation initiationcodon, and thus including nucleotides between the 5′ cap site and thetranslation initiation codon of an mRNA or corresponding nucleotides onthe gene; and the 3′ untranslated region (3′UTR), the portion of an mRNAin the 3′ direction from the translation termination codon, and thusincluding nucleotides between the translation termination codon and 3′end of an mRNA or corresponding nucleotides on the gene.

Antisense or sense oligonucleotides further comprise oligonucleotideshaving modified sugar-phosphodiester backbones (or other sugar linkages,such as those described in WO 91/06629) and wherein such sugar linkagesare resistant to endogenous nucleases. Such oligonucleotides withresistant sugar linkages are stable in vivo (i.e., capable of resistingenzymatic degradation) but retain sequence specificity to be able tobind to target nucleotide sequences. Oligonucleotides having modifiedbackbones include those that retain a phosphorus atom in the backboneand those that do not have a phosphorus atom in the backbone. In otherembodiments, both the sugar and the internucleoside linkage, i.e., thebackbone, of the nucleotide units are replaced with different groups. Afurther modification includes Locked Nucleic Acids (LNAs) in which the2′-hdroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ringto form a bycyclic sugar moiety. The sense or antisense oligonucleotidemolecules may also include nucleobase (base) modifications orsubstitutions.

The antisense and sense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare modified oligonucleotides. The compoundsof the invention may also be admixed, encapsulated, conjugated orotherwise associated with other molecules, molecule structures ormixtures of compounds, as for example, liposomes, receptor targetedmolecules, oral, rectal, topical or other formulations, for assisting inuptake, distribution and/or absorption.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell. Alternatively, a sense or an antisenseoligonucleotide may be introduced into a cell containing the targetnucleic acid sequence by formation of an oligonucleotide-lipid complex,as described in WO 90/10448. The sense or antisenseoligonucleotide-lipid complex is preferably dissociated within the cellby an endogenous lipase.

In another embodiment, the invention provides small molecules, whichbind, preferably specifically, to one or more of the cytokine receptorpolypeptides disclosed herein. In preferred embodiments, the smallmolecule is a small organic molecule, including small organic moleculesknown in the art as being an agonist or antagonist of a polypeptidecorresponding to a cytokine receptor disclosed herein.

In some embodiments, the small molecule is conjugated to a growthinhibitory agent or cytotoxic agent such as a toxin. Such toxinsinclude, for example, a maytansinoid or calicheamicin, an antibiotic, aradioactive isotope, a nucleolytic enzyme, or the like. The smallmolecules that find use in the therapeutic methods of the instantinvention preferably induce death of a cell to which they bind. Asdetailed above, binding to HTCs of myeloid origin may occur by virtue ofbinding to a surface-expressed marker disclosded herein; or by virtue ofbinding to the surface-expressed marker, which itself is bound to itsrecepetor on myelogenous HTCs.

7. PHARMACEUTICAL COMPOSITIONS

In the preparation of pharmaceutical compositions comprising theantibodies and/or antisense molecules and/or small molecule agonists orantagonists described in the teachings herein, a variety of vehicles andexcipients and routes of administration may be used, as will be apparentto the skilled artisan. Representative formulation technology is taughtin, inter alia, Remington: The Science and Practice of Pharmacy, 19thEd., Mack Publishing Co., Easton, Pa. (1995) and Handbook ofPharmaceutical Excipients, 3rd Ed, Kibbe, A. H. ed., Washington D.C.,American Pharmaceutical Association (2000); hereby incorporated byreference in their entirety.

The pharmaceutical compositions will generally comprise apharmaceutically acceptable carrier and a pharmacologically effectiveamount of the antibodies, or mixture of antibodies, or suitable saltsthereof. Use of a mixture of monoclonal antibodies specific to aprogenitor cell population as a therapeutic has a number of advantages.Abnormally proliferating cells have a tendency to mutate, and thus maylose the antigen recognized by a single type of monoclonal antibody.Moreover, antigen density of a single target antigen in the targetedcell could be low such that there is insufficient triggering of thesignals necessary for destruction of the cell by the immune system. Thepresent disclosure addresses these issues by providing multiple cytokinereceptors associated with HTCs of myeloid origin that can be targeted bya mixture of different monoclonal antibodies.

For known small molecule agonists or antagonists, pharmaceuticalcompositions can similarly be prepared based on known characteristics ofthe molecules.

The pharmaceutical composition may be formulated as powders, granules,solutions, suspensions, aerosols, solids, pills, tablets, capsules,gels, topical crèmes, suppositories, transdermal patches, and otherformulations known in the art.

For the purposes described herein, pharmaceutically acceptable salts ofthe antibodies is intended to include any art recognizedpharmaceutically acceptable salts including organic and inorganic acidsand/or bases. Examples of salts include sodium, potassium, lithium,ammonium, calcium, as well as primary, secondary, and tertiary amines,esters of lower hydrocarbons, such as methyl, ethyl, and propyl. Othersalts include organic acids, such as acetic acid, propionic acid,pyruvic acid, maleic acid, succinic acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, salicylic acid, etc.

As used herein, “pharmaceutically acceptable carrier” comprises anystandard pharmaceutically accepted carriers known to those of ordinaryskill in the art in formulating pharmaceutical compositions. Thus, theantibodies, by themselves, such as being present as pharmaceuticallyacceptable salts, or as conjugates, may be prepared as formulations inpharmaceutically acceptable diluents; for example, saline, phosphatebuffer saline (PBS), aqueous ethanol, or solutions of glucose, mannitol,dextran, propylene glycol, oils (e.g., vegetable oils, animal oils,synthetic oils, etc.), microcrystalline cellulose, carboxymethylcellulose, hydroxylpropyl methyl cellulose, magnesium stearate, calciumphosphate, gelatin, polysorbate 80 or the like, or as solid formulationsin appropriate excipients.

The pharmaceutical compositions will often further comprise one or morebuffers (e.g., neutral buffered saline or phosphate buffered saline),carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol,proteins, polypeptides or amino acids such as glycine, antioxidants(e.g., ascorbic acid, sodium metabisulfite, butylated hydroxytoluene,butylated hydroxyanisole, etc.), bacteriostats, chelating agents such asEDTA or glutathione, adjuvants (e.g., aluminium hydroxide), solutes thatrender the formulation isotonic, hypotonic or weakly hypertonic with theblood of a recipient, suspending agents, thickening agents and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilizate.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the compositions of this invention, the type ofcarrier will typically vary depending on the mode of administration.Antibody compositions may be formulated for any appropriate manner ofadministration, including for example, oral, nasal, mucosal,intravenous, intraperitoneal, intradermal, subcutaneous, andintramuscular administration.

For parenteral administration, the compositions can be administered asinjectable dosages of a solution or suspension of the substance in aphysiologically acceptable diluent with a pharmaceutical carrier thatcan be a sterile liquid such as sterile pyrogen free water, oils,saline, glycerol, polyethylene glycol or ethanol. Additionally,auxiliary substances, such as wetting or emulsifying agents,surfactants, pH buffering substances and the like can be present incompositions. Other components of pharmaceutical compositions are thoseof petroleum, animal, vegetable, or synthetic origin, for example,non-aqueous solutions of peanut oil, soybean oil, corn oil, cottonseedoil, ethyl oleate, and isopropyl myristate. Antibodies can beadministered in the form of a depot injection or implant preparationwhich can be formulated in such a manner as to permit a sustainedrelease of the active ingredient. An exemplary composition comprisesantibody at 5 mg/ml, formulated in aqueous buffer consisting of 50 mML-histidine, 150 mM NaCl, adjusted to pH 6.0 with HCl.

Typically, the compositions are prepared as injectables, either asliquid solutions or suspensions; solid or powder forms suitable forreconstitution with suitable vehicles, including by way example and notlimitation, sterile pyrogen free water, saline, buffered solutions,dextrose solution, etc., prior to injection. The preparation also can beemulsified or encapsulated in liposomes or micro particles such aspolylactide, polyglycolide, or copolymers, as further discussed below(see, e.g., Langer, Science 249:1527 (1990) and Hanes, Advanced DrugDelivery Rev. 28:97-119 (1997)).

Additionally, the compositions may also be introduced or encapsulatedinto the lumen of liposomes for delivery and for extending their lifetime ex vivo or in vivo. As known in the art, liposomes can becategorized into various types: multilamellar (MLV), stableplurilamellar (SPLV), small unilamellar (SUV) or large unilamellar (LUV)vesicles. Liposomes can be prepared from various lipid compounds, whichmay be synthetic or naturally occurring, including phosphatidyl ethersand esters, such as phosphotidylserine, phosphotidylcholine,phosphatidyl ethanolamine, phosphatidylinositol,dimyristoylphosphatidylcholine; steroids such as cholesterol;cerebrosides; sphingomyelin; glycerolipids; and other lipids (see, e.g.,U.S. Pat. No. 5,833,948).

Cationic lipids are also suitable for forming liposomes. Generally, thecationic lipids have a net positive charge and have a lipophilicportion, such as a sterol or an acyl or diacyl side chain. Preferably,the head group is positively charged. Typical cationic lipids include1,2-dioleyloxy-3-(trimethylamino)propane;N-[1-(2,3,-ditetradecycloxy)propyl]-N,N-dimethyl-N-N-hydroxyethylammoniumbromide; N-[1-(2,3-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide;N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride;3-[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol; anddimethyldioctadecylammonium.

Liposomes also include vesicles derivatized with a hydrophilic polymer,as provided in U.S. Pat. Nos. 5,013,556 and 5,395,619, herebyincorporated by reference, (see also, Kono, K. et al., J. ControlledRelease 68: 225-35 (2000); Zalipsky, S. et al., Bioconjug. Chem. 6:705-708 (1995)) to extend the circulation lifetime in vivo. Hydrophilicpolymers for coating or derivation of the liposomes include polyethyleneglycol, polyvinylpyrrolidone, polyvinylmethyl ether, polyaspartamide,hydroxymethyl cellulose, hydroxyethyl cellulose, and the like.

Liposomes are prepared by ways well known in the art (see, e.g., Szoka,F. et al., Ann. Rev. Biophys. Bioeng. 9: 467-508 (1980)). One typicalmethod is the lipid film hydration technique in which lipid componentsare mixed in an organic solvent followed by evaporation of the solventto generate a lipid film. Hydration of the film in aqueous buffersolution, preferably containing the subject antibodies, results in anemulsion, which is sonicated or extruded to reduce the size andpolydispersity. Other methods include reverse-phase evaporation (see,e.g., Pidgeon, C. et al., Biochemistry 26: 17-29 (1987); Duzgunes, N. etal., Biochim. Biophys. Acta. 732: 289-99 (1983)), freezing and thawingof phospholipid mixtures, and ether infusion.

In another embodiment, the carriers are in the form of microparticles,microcapsules, micropheres and nanoparticles, which may be biodegradableor non-biodegradable (see, e.g., “Microencapsulates: Methods andIndustrial Applications,” in Drugs and Pharmaceutical Sciences, Benita,S. ed, Vol 73, Marcel Dekker Inc., New York (1996); incorporated hereinby reference). As used herein, microparticles, microspheres,microcapsules and nanoparticles mean a particle, which is typically asolid, containing the substance to be delivered. The substance is withinthe core of the particle or attached to the particle's polymer network.Generally, the difference between microparticles (or microcapsules ormicrospheres) and nanoparticles is one of size. As used herein,microparticles have a particle size range of about 1 to about >1000microns. Nanoparticles have a particle size range of about 10 to about1000 nm.

A variety of materials are useful for making microparticles.Non-biodegradable microcapsules and microparticles include, but notlimited to, those made of polysulfones, poly(acrylonitrile-co-vinylchloride), ethylene-vinyl acetate,hydroxyethylmethacrylate-methyl-methacrylate copolymers. These areuseful for implantation purposes where the encapsulated compositiondiffuses out from the capsules. In another aspect, the microcapsules andmicroparticles are based on biodegradable polymers, preferably thosethat display low toxicity and are well tolerated by the immune system.These include protein based microcapsulates and microparticles made fromfibrin, casein, serum albumin, collagen, gelatin, lecithin, chitosan,alginate or poly-amino acids such as poly-lysine. Biodegradablesynthetic polymers for encapsulating may comprise polymers such aspolylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide)(PLGA), poly(caprolactone), polydioxanone trimethylene carbonate,polyhybroxyalkonates (e.g., poly(β-hydroxybutyrate)), poly(γ-ethylglutamate), poly(DTH iminocarbony (bisphenol A iminocarbonate), poly(ortho ester), and polycyanoacrylate. Various methods for makingmicroparticles containing the subject compositions are well known in theart, including solvent removal process (see, e.g., U.S. Pat. No.4,389,330); emulsification and evaporation (Maysinger, D. et al., Exp.Neuro. 141: 47-56 (1996); Jeffrey, H. et al., Pharm. Res. 10: 362-68(1993)), spray drying, and extrusion methods.

Another type of carrier is nanoparticles, which are generally suitablefor intravenous administrations. Submicron and nanoparticles aregenerally made from amphiphilic diblock, triblock, or multiblockcopolymers as is known in the art. Polymers useful in formingnanoparticles include, but are limited to, poly(lactic acid) (PLA; seeZambaux et al., J. Control Release 60: 179-188 (1999)),poly(lactide-co-glycolide), blends of poly(lactide-co-glycolide) andpolycarprolactone, diblock polymer poly(l-leucine-block-l-glutamate),diblock and triblock poly(lactic acid) (PLA) and poly(ethylene oxide)(PEO) (De Jaeghere, F. et al., Pharm. Dev. Technol.; 5: 473-83 (2000)),acrylates, arylamides, polystyrene, and the like. As described formicroparticles, nanoparticles may be non-biodegradable or biodegradable.Nanoparticles may be also be made from poly(alkylcyanoacrylate), forexample poly(butylcyanoacrylate), in which the therapeutic compositionis absorbed onto the nanoparticles and coated with surfactants (e.g.,polysorbate 80). Methods for making nanoparticles are similar to thosefor making microparticles and include, among others, emulsionpolymerization in continuous aqueous phase, emulsification-evaporation,solvent displacement, and emulsification-diffusion techniques (see,e.g., Kreuter, J. Nano-particle Preparation and Applications, inMicrocapsules and Nanoparticles in Medicine and Pharmacy, pg. 125-148,(M. Donbrow, ed.) CRC Press, Boca Rotan, Fla. (1991); incorporatedherein by reference).

The pharmaceutical compositions described herein may be presented inunit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are typically sealed in such a way to preserve thesterility and stability of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles, as indicated above. Alternatively, apharmaceutical composition may be stored in a freeze-dried conditionrequiring only the addition of a sterile liquid carrier immediatelyprior to use.

8. USE OF ANTIBODIES AND OTHER AGENTS 8.1 Therapeutic Use of Antibodiesand Small Molecules

Methods of immunotargeting cancer cells using antibodies or antibodyfragments are well known in the art. U.S. Pat. No. 6,306,393 describesthe use of anti-CD22 antibodies in the immunotherapy of B-cellmalignancies, and U.S. Pat. No. 6,329,503 describes immunotargeting ofcells that express serpentine transmembrane antigens. Antibodiesdescribed herein (including humanized or human monoclonal antibodies orfragments or other modifications thereof, optionally conjugated tocytotoxic agents) can be introduced into a patient such that theantibody binds to cancer cells or their secreted expression products andmediates the destruction of the cells and the tumor and/or inhibits thegrowth of the cells or the tumor. Without intending to limit thedisclosure, mechanisms by which such antibodies can exert a therapeuticeffect may include complement-mediated cytolysis, antibody-dependentcellular cytotoxicity (ADCC), modulating the physiologic function of thetumor antigen, inhibiting binding or signal transduction pathways,modulating tumor cell differentiation or proliferation, altering tumorangiogenesis factor profiles, modulating the secretion of immunestimulating or tumor suppressing cytokines and growth factors,modulating cellular adhesion, and/or by inducing apoptosis. Theantibodies can also be conjugated to toxic or other therapeutic agents,such as radioligands or cytosolic toxins, discussed in detail above, andmay also be used therapeutically to deliver the toxic or therapeuticagent directly to tumor cells.

In preferred embodiments, the compositions have applications to thetreatment of conditions or diseases involving myeloid cells of thehematopoietic system. The present disclosure further provides methods ofusing the antibodies to target myeloid leukemic stem cells. For example,the disclosure provides methods of using the antibodies to treatdisorders involving cells of the myeloid lineages. Various diseases haveorigins in the committed progenitor cell populations, or involveprogenitor cells by differentiation of diseased cells through themyeloid pathway.

By “treatment” herein is meant therapeutic or prophylactic treatment, ora suppressive measure for the disease, disorder or undesirablecondition. Treatment encompasses administration of the subjectantibodies in an appropriate form prior to the onset of disease symptomsand/or after clinical manifestations, or other manifestations, of thedisease to reduce disease severity, halt disease progression, oreliminate the disease. Prevention of the disease includes prolonging ordelaying the onset of symptoms of the disorder or disease, preferably ina subject with increased susceptibility to the disease.

The antibodies described herein are particularly applicable to thetreatment of myeloproliferative disorders, also referred to generally ashematopoietic malignancies, which are proliferative disorders involvingcells of the myeloid lineage. The term malignancy refers to growth andproliferation of one or more clones of abnormal cells. Leukemiatypically describes a condition in which abnormal cells are present inthe bone marrow and peripheral blood. Myeloproliferative disorders, alsocalled myeloid leukemias or myelogenous leukemias, are categorized intothree general groups of conditions: dysmyelopoietic disorder, acutemyeloproliferative leukemia, and chronic myeloproliferative disorder.

Myelodysplastic Syndromes (MDS) include a group of closely-related bloodformation disorders, in which the bone marrow shows qualitative andquantitative changes suggestive of a preleukemic process, but having achronic course that does not necessarily terminate as acute leukemia. Avariety of terms, including preleukemia, refractory anemia, refractorydysmyelopoietic anemia, smoldering or subacute leukemia, dysmyelopoieticsyndrome (DMPS), and myelodysplasia, have all been used to describe MDS.These conditions are all characterized by a cellular marrow withimpaired maturation (dysmyelopoiesis) and a reduction in the number ofblood cells. DMPS is characterized by presence of megablastoids,megarkaryocyte dysplasia, and an increase in number of abnormal blastcells, reflective of enhanced granulocyte maturation process. Patientswith DMPS show chromosomal abonormalities similar to those found inacute myeloid leukemia and progress to acute myeloid leukemia in acertain fraction of afflicted patients (Kardon, N. et al., Cancer50(12):2834-2838 (1982)).

Acute myeloproliferative leukemia (AML), also known as acutenonlymphocytic leukemia, acute myelocytic leukemia, acute myeloblasticleukemia, and acute granulocytic leukemia, is characterized by thepresence of abnormal hematopoietic progenitor cells that have beenblocked at an undifferentiated or partially differentiated stage ofmaturation, and thus are unable to differentiate into myeloid,erythroid, and/or megakaryocytic cell lines. The abnormal cells blockdifferentiation of normal progenitor cells in the bone marrow, resultingin thrombocytopenia, anaemia, and granulocytopenia. Diagnosis of AML ismade when at least 30% of nucleated cells in the bone marrow are blasts.Acute myeloid leukemia is further divided into subtypes M1 to M7 basedon morphology of the proliferating cells and cytochemical stainingproperties.

Chronic myeloproliferative disorders are a collection of conditionscharacterized by increased number of mature and immature granulocytes,erythrocytes, and platelets. Chronic myeloproliferative disorders cantransition to other forms within this group, with a tendency toterminate in acute myeloid leukemia. Specific diseases within this groupinclude polycythemia vera, chronic myeloid leukemia, agnogenic myeloidleukemia, essential thrombocythemia, and chronic neutrophilic leukemia.

The therapeutic preparations can use non-modified marker specificantibodies or antibodies conjugated with a therapeutic compound, such asa toxin or cytotoxic molecule, depending on the functionality of theantibody. Generally, when non-modified antibodies are used, they willtypically have a functional Fc region. By “functional Fc region” hereinis meant a minimal sequence for effecting the biological function of Fc,such as binding to Fc receptors, particularly FcγR (e.g., FcγRI, FcγRII,and FcγRIII). Without being bound by theory, it is believed that the Fcregion may affect the effectiveness of anti-tumor monoclonal antibodiesby binding to Fc receptors immune effector cells and modulating cellmediated cytotoxicity, endocytosis, phagocytosis, release ofinflammatory cytokines, complement mediated cytotoxicity, and antigenpresentation. In this regard, polyclonal antibodies, or mixtures ofmonoclonals, will be advantageous because they will bind to differentepitopes (of a single antigen or of different antigens corresponding todifferent cytokine receptors) and thus have a higher density of Fc onthe cell surface as compared to when a single monoclonal antibody isused. Of course, to enhance their effectiveness in depleting targetedcells, or where non-modified antibodies are not therapeuticallyeffective, antibodies conjugated to toxins or cytotoxic agents may beused. Thus, not only are the antibodies useful as therapeutic moleculesthemselves, they also find utility in targeted delivery of therapeuticmolecules to myeloid cells.

Alternatively, where the antibodies exhibit a direct effect on antigenand/or cell function, enhancement of the Fc receptor functionality maybe less significant. For example, this may be the case where binding ofthe marker specific antibody sterically inhibits interaction betweenantigen and its corresponding ligand.

The antibody compositions may be used either alone or in combinationwith other therapeutic agents to increase efficacy of traditionaltreatments for myeloid leukemias, or to target abnormal cells nottargeted by the antibodies. Combining the antibody therapy method with achemotherapeutic, radiation or surgical regimen may be preferred inpatients that have not received chemotherapeutic treatment, whereastreatment with the antibody therapy may be indicated for patients whohave received one or more chemotherapies. Additionally, antibody therapycan also enable the use of reduced dosages of concomitant chemotherapy,particularly in patients that do not tolerate the toxicity of thechemotherapeutic agent very well. Furthermore, antibody treatment ofcancer patients with tumors resistant to chemotherapeutic agents mightinduce sensitivity and responsiveness to these agents in combination.

In one aspect, the antibodies are used adjunctively with therapeuticcytotoxic agents, including, by way of example and not limitation,busulfan, thioguanine, idarubicin, cytosine arabinoside,6-mercaptopurine, doxorubicin, daunorubicin, etoposide, and hydroxyurea.Other agents useful as adjuncts to antibody therapy are compoundsdirected specifically to an abnormal cellular molecule found in thedisease state. These agents can be disease specific. For example, fortreating chronic myeloid leukemia arising from BCR-ABL activity, oneclass of useful compounds are inhibitors of abl kinase activity, such asImatinib, an inhibitor of bcr-abl kinase, and antisense oligonucleotidesagainst bcr (e.g., Oblimersen). Other agents include, among others,interferon-alpha, humanized anti-CD52, deacetylase inhibitor FR901228(depsipeptide), and the like.

In another aspect, isotopes are attached to the antibodies and/orfragments for therapeutic purposes. By “isotope” is meant atoms with thesame number of protons and hence of the same element but with differentnumbers of neutrons (e.g., ¹H vs. ²H or D). The term “isotope” includes“stable isotopes”, e.g. non-radioactive isotopes, as well as“radioactive isotopes”, e.g. those that decay over time, and radioactiveradionuclides. In one embodiment, the antibodies and/or fragments arelabeled with a radioisotope, which are useful in radioimmunotherapy.Suitable radioisotopes include without limitation an alpha-emitter, abeta-emitter, and an Auger electron-emitter (Behrt, T. et al., (2000).Eur. J. Nuclear Med. vol. 27 (7):753-765; Vallabhajosula, S. et al., J.Nucl. Med. (2005) April; 46(4):634-41). Such radioisotopes includewithout limitation [65]Zinc, [140]neodymium, [177]lutetium,[179]lutetium, [176m]lutetium, [67]gallium, [159]gallium, [161]terbium,[153]samarium, [169]erbium, [175]ytterbium, [161]holmium, [166]holmium,[167]thulium, [142]praseodymium, [143]praseodymium, [145]praseodymium,[149]promethium, [150]europium, [165]dysprosium, [111]indium,[131]iodine, [125]iodine, [123]iodine, [88]yttrium and [90]yttrium.Suitable radioactive radionuclides include without limitation, ⁶⁷Cu,⁹⁰Y, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, ²¹²Bi. These and other uses of theantibodies will be apparent to those of ordinary skill in the art inlight of the disclosures provided herein.

Known small molecule agonists or antagonists can also find use inmethods of treating of one or more of the myelogenous hematologicalproliferative disorders described above. Generally, the methods compriseadministration of a therapeutically effective amount of the smallmolecule (or mixture of small molecules). Similarly as detailed abovewith respect to immunotherapies, the therapeutic composition can use thenon-modified small molecule or, optionally, the small molecule isconjugated to a toxin or cytotoxic molecule or growth inhibitory agent.For example, the small molecule may be conjugated to a maytansinoid orcalicheamicin, an antibiotic, a radioactive isotope, a nucleolyticenzyme, or the like.

Binding of the small molecule to a HTC of myeloid origin preferablyinduce cell death. Cell death may be mediated by the conjugatedcytotoxic molecule or by the physiological response induced by thebinding of the small molecule itself.

8.1.1. Administration and Dosages

The amount of the compositions needed for achieving a therapeutic effectwill be determined empirically in accordance with conventionalprocedures for the particular purpose. Generally, for administering thecompositions ex vivo or in vivo for therapeutic purposes, thecompositions are given at a pharmacologically effective dose. By“pharmacologically effective amount” or “pharmacologically effectivedose” is meant an amount sufficient to produce the desired physiologicaleffect or amount capable of achieving the desired result, particularlyfor treating or retreating the disorder or disease condition, includingreducing or eliminating one or more symptoms or manifestations of thedisorder or disease. As an illustration, administration of antibodies toa patient suffering from a myeloproliferative disorder provides atherapeutic benefit not only when the underlying disease is eradicatedor ameliorated, but also when the there is a decrease in the severity orduration of the symptoms associated with the disease. Therapeuticbenefit also includes halting or slowing the progression of theunderlying disease or disorder, regardless of whether improvement isrealized.

The amount administered to the subject will vary depending upon theagent being administered, the purpose of the administration, such asprophylaxis or therapy, the state of the subject, the manner ofadministration, the number of administrations, the intervals betweenadministrations, and the like. These can be determined empirically bythose skilled in the art and may be adjusted based on the extent of thetherapeutic response. Factors to consider in determining an appropriatedose include, but are not limited to, size and weight of the subject,the age and sex of the subject, the severity of the symptoms, the stageof the disease, method of delivery of the agents, half-life of theagents, efficacy of the agents, and what other therapy regimes have beenor will be administered. Stage of the disease to consider includeswhether the disease is acute or chronic, relapsing or remitting phase,as well as the progressiveness of the disease. Determining the dosagesand times of administration for a therapeutically effective amount arewell within the skill of the ordinary person in the art, in light of thedisclosures provided herein.

For any compositions of the present disclosure, the therapeuticallyeffective dose is readily determined by methods well known in the art.For example, an initial effective dose can be estimated from cellculture or other in vitro assays. For example, Sliwkowsky, M. X. et al.,Semin. Oncol. 26(suppl. 12) 60-70 (1999) describes in vitro measurementsof antibody dependent cellular cytoxicity. A dose can then be formulatedin animal models to generate a circulating concentration or tissueconcentration, including that of the IC₅₀ as determined by the cellculture assays. A suitable animal model for leukemia is described indetail in the Examples below.

In addition, the toxicity and therapeutic efficacy are generallydetermined by cell culture assays and/or experimental animals, typicallyby determining a LD₅₀ (lethal dose to 50% of the test population) andED₅₀ (therapeutically effectiveness in 50% of the test population). Thedose ratio of toxicity and therapeutic effectiveness is the therapeuticindex. Preferred are compositions, individually or in combination,exhibiting high therapeutic indices. Determination of the effectiveamount is well within the skill of those in the art, particularly giventhe detailed disclosure provided herein. Guidance is also found instandard reference works, for example Fingl and Woodbury, GeneralPrinciples In: The Pharmaceutical Basis of Therapeutics pp. 1-46 (1975),and the references cited therein.

To achieve an initial tolerizing dose, consideration is given to thepossibility that the antibodies may be immunogenic in humans and innon-human primates. The immune response may be biologically significantand may impair the therapeutic efficacy of the antibody even if theantibody is partly or chiefly comprised of human immunoglobulinsequences such as, for example, in the case of a chimeric or humanizedantibody. Within certain embodiments, an initial high dose of antibodyis administered such that a degree of immunological tolerance to thetherapeutic antibody is established. The tolerizing dose is sufficientto prevent or reduce the induction of an antibody response to repeatadministration of the marker specific antibody. Preferred ranges for thetolerizing dose are between 10 mg/kg body weight to 50 mg/kg bodyweight, inclusive. More preferred ranges for the tolerizing dose arebetween 20 and 40 mg/kg, inclusive. Still more preferred ranges for thetolerizing dose are between 20 and 25 mg/kg, inclusive.

Within these therapeutic regimens, the therapeutically effective dose ofantibodies is preferably administered in the range of 0.1 to 10 mg/kgbody weight, inclusive. More preferred second therapeutically effectivedoses are in the range of 0.2 to 5 mg/kg body weight, inclusive. Stillmore preferred therapeutically effective doses are in the range of 0.5to 2 mg/kg, inclusive. Within alternative embodiments, the subsequenttherapeutic dose or doses may be in the same or different formulation asthe tolerizing dose and/or may be administered by the same or differentroute as the tolerizing dose.

For the purposes of this invention, the methods of administration arechosen depending on the condition being treated, the form of the subjectantibodies or other agents, and the pharmaceutical composition.Administration of the antibody compositions or small moleculecompositions can be done in a variety of ways, including, but notlimited to, continuously, subcutaneously, intravenously, orally,topically, transdermal, intraperitoneal, intramuscularly, andintravesically. For example, microparticle, microsphere, andmicroencapsulate formulations are useful for oral, intramuscular, orsubcutaneous administrations. Liposomes and nanoparticles areadditionally suitable for intravenous administrations. Administration ofthe pharmaceutical compositions may be through a single route orconcurrently by several routes. For instance, intraperitonealadministration can be accompanied by intravenous injections. Preferablythe therapeutic doses are administered intravenously, intraperitonealy,intramuscularly, or subcutaneously. In some embodiments, the smallmolecule compositions can be administered orally

The compositions may be administered once or several times. In someembodiments, the compositions may be administered once per day, a few orseveral times per day, or even multiple times per day, depending upon,among other things, the indication being treated and the judgement ofthe prescribing physician.

Administration of the compositions may also be achieved throughsustained release or long-term delivery methods, which are well known tothose skilled in the art. By “sustained release or” “long term release”as used herein is meant that the delivery system administers apharmaceutically therapeutic amount of subject agents for more than aday, preferably more than a week, and most preferable at least about 30days to about 60 days, or longer. Long term release systems may compriseimplantable solids or gels containing the antibodies, such asbiodegradable polymers described above (Brown, D. M. et al., AnticancerDrugs 7: 507-513 (1996)); pumps, including peristaltic pumps andfluorocarbon propellant pumps; osmotic and mini-osmotic pumps; and thelike.

The method of the invention contemplates the administration of markerspecific monoclonal antibodies, as well as combinations of differentmAbs. As discussed above, two or more monoclonal antibodies may providean improved effect compared to a single antibody. For example, acombination of a monoclonal antibody with another monoclonal antibodythat binds a different antigen, e.g., an antigen corresponding to adifferent cytokine receptor, may provide an improved effect compared toa single antibody. Such mAb “cocktails” may have certain advantages inas much as they contain mAbs, which exploit different effectormechanisms or combine directly cytotoxic mAbs with mAbs that rely onimmune effector functionality. Specific mAbs in combination may exhibitsynergistic therapeutic effects. Some methods of the invention alsocontemplate adminisration of one or more known small molecule agonistsor antagonists, alone or in combination with one or more mAbs describedherein. The small molecule in combination with specific mAbs may exhibitsynergistic therapeutic effects.

8.2 Diagnostic Use of Antibodies and Other Agents

The present invention further provides methods to identify the presenceof an antigen using the compositions of the present invention,optionally conjugated or otherwise associated with a suitable label.Such methods comprise incubating a test sample with one or more of themarker specific antibodies of the present invention and assaying forantibodies that bind to components within the test sample. Conditionsfor incubating the antibody with a test sample may vary. Incubationconditions depend on the format employed, the detection methodsemployed, and the antibody used in the assay. One skilled in the artwill recognize that any one of the commonly available immunologicalassay formats can readily be adapted to employ antibodies of the presentinvention (see Chard, T., An Introduction to Radioimmunoassay andRelated Techniques, Elsevier Science Publishers, Amsterdam, TheNetherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985).

The sample to be assessed can be any sample that contains an expressionproduct (e.g., RNA transcript or extracellular protein). A “test sample”generally refers to a sample obtained or derived from a patientafflicted with, or suspected of being afflicted with, a hematologicalproliferative disorder of myeloid origin. The test samples of thepresent invention include cells, protein or membrane extracts of cells,or biological fluids. Samples can comprise brain, blood, serum, plasma,lymphatic fluid, bone marrow, plasma, lymph, urine, tissue, mucus,sputum, saliva or other cell samples. The test sample used will varybased on the assay format, nature of the detection method and thetissues, cells or extracts used and the sample to be assayed. Methodsfor preparing protein extracts or membrane extracts of cells are wellknown in the art and can be readily adapted in order to obtain a samplewhich is compatible with the system utilized.

In preferred embodiments, the test sample is obtained from peripheralblood, particularly from mobilized peripheral blood (MPB). The sampleinitially obtained from a subject is generally enriched for stem cells.Hematopoietic stem cells can be identified by the presence or absence ofcertain surface markers, as described above, including, e.g., CD34⁺;CD38⁻; as well as Lin⁻ and/or CD90⁺.

The present invention provides diagnostic methods for hematologicalproliferative disorders of myeloid origin, where the level of anexpression product of one or more of the disclosed cytokine receptors isdetected. “Detected,” and its grammatical variations, refers toassessing, measuring, reading, or otherwise determining a value for thelevel or amount of expression product corresponding to one of more ofthe cytokine receptors disclosed herein. An expression product“corresponds to” a designated cytokine receptor when it is derivedtherefrom via transcription and/or translation of the gene encoding thedesignated cytokine receptor. Expression levels refer to the amount ofexpression of the expression product, as described herein. A value foran expression level is also referred to as a “signal.” The values forexpression levels can be absolute or relative values, e.g., valuesprovided in comparison to control levels. The values for expressionlevels can be raw values, or values that are optionally rescaled,filtered and/or normalized. The approach used will depend, for example,on the nature of the expression product (e.g., RNA or polypeptide) aswell as specific characteristics of the product, and the intended usefor the data.

For example, in one embodiment, the expression product is atranscription product, such as RNA. RNA includes, e.g, mRNA rRNA, tRNA,snRNA, and the like, inclulding any nucleic acid molecule that istranscribed from a gene. The nucleic acid molecule levels measured canbe derived directly from the gene or, alternatively, from acorresponding regulatory gene or regulatory sequence element.Additionally, variants of genes and gene expression products including,for example, spliced variants and polymorphic alleles, can be detected.

Methods of detecting the level of an RNA transcript include, forexample, utilizing a specific hybridization probe or an array of suchprobes. In a preferred embodiment, the probe comprises a polynucleotidesequence that can hybridize to all or a portion of the transcribed RNAsequence.

The stringency conditions allowing hybridization can be high to moderateto low. As used herein, conditions of moderate stringency refer to thoseknown to the ordinarily skilled artisian, e.g., as defined by Sambrooket al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1, pp.1.101-104, Cold Spring Harbor Laboratory Press, (1989). Moreratestringency conditions include use of a prewashing solution fornitrocellulose filters 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0),hybridization conditions of 50% formamide, 6×SSC at 42° C. (or othersimilar hybridization solution, such as Stark's solution, in 50%formamide at 42° C.), and washing conditions of about 60° C., 0.5×SSC,0.1% SDS. High stringency conditions usually involve, for example,hybridization conditions as above, with washing at 68° C., 0.2×SSC, 0.1%SDS. The skilled artisan will recognize that the temperature and washsolution salt concentration can be adjusted as necessary according tofactors such as the length of the probe. The hybridization probe can beof any length and usually consists of at least about 5 nucleotides, atleast about 10, at least about 15, at least about 20, or at least about30 nucleotides. Longer lengths are suitable to lower stringencyconditions.

The probe may include natural (i.e. A, G, U, C, or T) or modified bases(7-deazaguanosine, inosine, etc.). In addition, the bases in probes maybe joined by a linkage other than a phosphodiester bond, so long as thebond does not interfere with hybridization. Thus, probes may be peptidenucleic acids in which the constituent bases are joined by peptide bondsrather than phosphodiester linkages.

In some embodiments, more than one hybridization probe is used, e.g.,two, three, four, five, 10 or more probes, up to, including and beyondas many probes as cytokine receptors disclosed herein. In someembodiments, different probes are directed to different RNA products,e.g., RNA products corresponding to two or more different cytokinereceptors disclosed herein. For example, probes for detecting theexpression level of two, three, four, five, or more of the cytokinereceptor disclosed herein may be used.

In preferred embodiments, the probes are immobilized, e.g., on an array,in different known locations. An array refers to a solid support with asurface to which a plurality of different nucleic acid sequences(probes) are attached. The array can be prepared either synthetically orbiosynthetically, and can assume a variety of formats, e.g., librariesof compounds tethered to resin beads, silica chips, or other solidsupports, as well as libraries of nucleic acids prepared by spottingnucleic acids onto a substrate. The solid support can be any material orgroup of materials having a rigid or semi-rigid surface or surfaces.Generally, at least one surface of the solid support will besubstantially flat, although in some case the array will include wells,raised regions, pins, etched trenches, or the like. Although a planararray surface is preferred, the array may be fabricated on a surface ofvirtually any shape or even a multiplicity of surfaces. Solid support(s)can also take the form of beads, resins, fibers such as fiber optics,gels, microspheres, or other geometric configurations. See U.S. Pat.Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, which arehereby incorporated in their entirety for all purposes.

Such arrays are generally termed “microarrays”, or colloquially “chips”,and have been described in the art, for example, U.S. Pat. Nos.5,143,854, 5,445,934, 5,744,305, 5,677,195, 6,040,193, 5,424,186 andFodor et al., Science, 251:767-777 (1991), each of which is incorporatedby reference in its entirety for all purposes. These arrays maygenerally be produced using mechanical synthesis methods or lightdirected synthesis methods, which can incorporate a combination ofphotolithographic methods and solid phase synthesis methods. Techniquesfor the synthesis of these arrays using mechanical synthesis methods aredescribed in, e.g., U.S. Pat. No. 5,384,261, incorporated herein byreference in its entirety for all purposes. Commercially availableprobes and arrays can be used, including, for example, Affymetrix human0133 Plus 2.0 Array.

In a preferred embodiment, the expression product is mRNA and the mRNAlevels are obtained by contacting the sample with a suitable microarray,and determining the extent of hybridization of the nucleic acid in thesample to the probes on the microarray.

For example, mRNA levels can be obtained from a GeneChip™ probe array orMicroarray (Affymetrix, Inc.) (U.S. Pat. Nos. 5,631,734, 5,874,219,5,861,242, 5,858,659, 5,856,174, 5,843,655, 5,837,832, 5,834,758,5,770,722, 5,770,456, 5,733,729, 5,556,752, all of which areincorporated herein by reference in their entirety), and the expressionlevels can be calculated with software (e.g., Affymetrix GENECHIP™software). Briefly, nucleic acids (e.g., mRNA) from a sample which hasbeen subjected to particular stringency conditions hybridize to theprobes on the chip. The nucleic acid to be analyzed (e.g., mRNAcorresponding to an HTC marker) is isolated, amplified and labeled witha detectable label, (e.g., ³²P or fluorescent label) prior tohybridization to the arrays. Once hybridization occurs, the arrays areinserted into a scanner which can detect patterns of hybridization. Thehybridization data are collected as light emitted from the labeledgroups, which are now bound to the probe array. The probes thatperfectly match the mRNA corresponding to an HTC marker produce astronger signal than those that have mismatches. Since the sequence andposition of each probe on the array are known, by complementarity, theidentity of the nucleic acid applied to the probe is determined.Quantitation from the hybridization of labeled mRNA/DNA microarray canbe performed by scanning the microarrays to measure the amount ofhybridization at each position on the microarray. This can be performedwith, for example, an Affymetrix scanner (Affymetrix, Santa Clara,Calif.). Microarrays are only one method of detecting RNA levels. Othermethods known in the art or developed in the future can be used with thepresent invention.

In another embodiment, the expression product is a translation product,e.g. one or more cytokine receptors disclosed herein. Cytokine receptorproteins include any polypeptide or derivative thereof, including, e.g.,peptides, glycoproteins, lipoproteins and nucleic acid-proteincomplexes.

Techniques for protein detection and quantitation are known in the art.For example, antibodies specific for the protein or polypeptide can beobtained using methods which are routine in the art, and the specificbinding of such antibodies to protein or polypeptide expression productscan be detected and measured. Methods of detecting the level of proteinspreferably involve utilizing antibodies of the instant disclosure, asdiscussed above. In some embodiments, small molecules known to bind to apolypeptide corresponding to a disclosed cytokine receptor can similarlybe detectably labeled and/or attached to a solid support.

In some embodiments, more than one antibody is used, e.g., two or moremAbs. In some embodiments, different mAbs are directed to aproteinaceous product expressed from two or more different cytokinereceptors disclosed herein. For example, antibodies for detecting theexpression level of two, three, four, five, or more of the differentcytokine receptors disclosed herein may be used. In some embodiments,both RNA and protein levels are detected in a given sample or multiplesamples from an individual.

The RNA or antigen level can be compared to control levels, e.g., levelsobtained using normal HSCs. A control sample comprising one or morenormal HSCs can be obtained, e.g., from an individual not afflicted witha hematological proliferative disorder, e.g, not afflicted with ahematological proliferative disorder of myeloid origin. Preferably, thecontrol sample is obtained in a manner similar to that used to obtainthe test sample, e.g, being obtained from the same organs, tissues orfluids (as detailed above with respect to test samples); and sorted toenrich for corresponding cell types. Similarly, the level of RNA orantigen preferably is assesed in the control sample in a manner similarto that used to obtain the test values. Methods are known in the art forpermitting direct comparison of test and control levels, and a specificexample of such comparison is detailed below in Example 1. In someembodiments, control levels are provided by previously-obtained data,such as from control samples that have been detected in prior assays;from published data; and/or from accessible data bases.

Diagnosis is based on a correlation between the level of a givenexpression product in a test sample compared to that in a controlsample. For example, as detailed in Example 1 below, mRNA levels of thecytokine receptors disclosed herein are higher in AML HTC samples ascompared to normal HSC samples. Preferably, the difference in expressionlevels is at least about 2 fold, at least about 3 fold, at least about 5fold, at least about 7 fold, at least about 10 fold or at least about 15fold. In particularly preferred embodiments, the difference inexpression levels is at least about 20 fold, at least about 30 fold, atleast about 40 fold or at least about 50 fold. In still more preferredembodiments, the difference in expression levels is at least about 70fold, at least about 100 fold, at least about 200, fold or as much asnearly 300 fold, 400 fold or 500 fold. For example, IL13RA1 mRNA levelsin AML HTCs is over 35 times that in normal HSCs (see Table 1).Accordinlgy, the information provided by the present disclosure, aloneor in conjunction with other test results, aids in sample classificationand diagnosis of hematological proliferative disorders of myeloidorigin, such as AML.

In some embodiments, more than one test sample and/or more than onecontrol sample are obtained and/or detected. For example, as illustratedin Example 1 below, all of 3 normal samples showed low levels ofexpression of each cytokine receptor, while at least 2 out of 3 AMLsamples showed high expression levels. Alternatively, AML samplesover-expressed the cytokine receptor by at least about 5 fold comparedto normal HSCs. Diagnosis may take into account the difference inexpression levels between more than one test sample and/or more than onecontrol sample. In some embodiments, repeat assays are performed using agiven sample.

In some embodiments, expression of more than one cytokine receptor canbe detected. The different cytokine receptors can be detectedsimultaneously. For example, in some embodiments, two, three, four,five, or more of the different cytokine receptors disclosed herein aredetected. The detection of numerous genes can provide a more accurateevaluation of the sample. The correlation between expression productlevels and a given disease, such as AML, can be determined using avariety of methods. Methods of classifying samples are described, forexample, in U.S. patent application Ser. No. 09/544,627, filed Apr. 6,2000 by Golub et al., the teachings of which are incorporated herein byreference in their entirety.

The present invention also provides prognostic methods for predictingthe efficacy of treating a haematological proliferative disorder ofmyeloid origin, where the level of an expression product of one or moreof the disclosed cytokine receptors is detected and wherein theexpression product level is correlated with a treatment outcome.“Treatment outcome” as used herein refers to the efficacy of a treatmentwith respect to a disease, that is, the response of the disease to aparticular treatment. The levels of expression products can be used toassess the likelihood that a given disorder will respond well to aparticular treatment, or to determine which of a number of treatmentoptions is more preferable. In some embodiments, the treatment is onedisclosed herein, e.g., one or more of the therapeutic uses of theantibodies described herein. In some embodiments, the treatment is atreatment otherwise known or used or to be used in the art; or acombination of such treatments with one or more of those disclosedherein.

The disclosure above regarding, for example, test and control samples;stem cell sorting; use of one or more cytokine receptors; detection ofRNA and/or antigen levels; correlation of data and so forth, as providedin the case of diagnostic methods, also applies to the prognosticmethods disclosed herein.

In a preferred embodiment, the expression product is RNA and RNA levelsin test samples are compared to control levels. In a paticularlypreferred embodiment, the expression product is mRNA and the mRNA levelsare obtained by contacting the sample with a suitable microarray, anddetermining the extent of hybridization of the nucleic acid in thesample to the probes on the microarray.

In particularly preferred embodiments, the expression product is mRNAand lower mRNA levels correlate with more favorable treatment outcomes.For example, a TRGV9 expression level that is about 10 times higher thancontrol levels indicates a more favorable outcome than where the TRGV9expression level is about 30 times higher. In some embodiments, theexpression levels of more than one cytokine receptor is be detected; andlower expression levels of multiple markers is further indication of amore favorable treatment outcome.

The present invention also provides methods for monitoring the efficacyof treating a haematological proliferative disorder of myeloid origin,where the level of an expression product of one or more of the disclosedcytokine receptors is detected at various time points and correlatedwith treatment outcome. The various time points can include, forexample, time of diagnosis, times prior to commencing a treatmentregime; times at intervals during a treatment regime; and times afterthe conclusion of treatment regime. The time intervals can includeseveral hours; a day; 2, 3, or 4 days; a week; 2, 3, 4, 5 or 6 weeks; amonth, 2, 3, 4, 5, or 6 months, a year, or several years post-initiationof a treatment regime. In some embodiments, the treatment is onedisclosed herein, e.g., one or more of the therapeutic uses of theantibodies described herein. In some embodiments, the treatment is atreatment otherwise known or used or to be used in the art; or acombination of such treatments with one or more of those disclosedherein.

The disclosure above regarding, for example, test and control samples;stem cell sorting; use of one or more cytokine receptors; detection ofRNA and/or protein levels; correlation of data and so forth, as providedin the case of diagnostic methods, also applies to the monitoringmethods disclosed herein.

In a preferred embodiment, the expression product is RNA and RNA levelsin test samples are compared to control levels. In a paticularlypreferred embodiment, the expression product is mRNA and the mRNA levelsare obtained by contacting the sample with a suitable microarray, anddetermining the extent of hybridization of the nucleic acid in thesample to the probes on the microarray.

Monitoring the efficacy of a treatment is useful, e.g., in facilitatingclinical management of the disease, e.g., where decisions must be madeas to whether to continue a treatment course or advance to othertreatment options. Whether a myeloid leukemia is responding positivelyto a treatment can be determined based on changes in the level of agiven expression product (or products) in test samples taken at varioustime points, e.g., before and after administration of a particulartreatment.

A shift in expression product levels from a level correlated with HTCsof myeloid origin towards a level correlated with normal HSCs isevidence of an effective therapeutic regime. In some embodiments, areduction in the expression product level corresponding to one or moreof the cytokine receptors disclosed herein indicates a positive responseto treatment. The reduction indicates a trend towards levels seen innormal HSC samples. For example, as detailed in Example 1 below, theexpression product level in AML HTCs is higher than that in normal HSCsfor each of the cytokine receptors disclosed herein. A reduction inexpression level in one or more of the disclosed cytokine receptors,e.g., in test samples obtained after the commencement of treatment,would indicate a positive response.

In preferred embodiments, the expression level for one or more HTCmarkers is reduced at least about 5 fold, at least about 7 fold, atleast about 10 fold or at least about 15 fold, during the course oftreatment. In particularly preferred embodiments, the expression levelfor one or more HTC markers is reduced at least about 20 fold, at leastabout 30 fold, at least about 40 fold or at least about 50 fold, duringthe course of treatment. In still more preferred embodiments, theexpression level for one or more HTC markers is reduced at least about70 fold, at least about 100 fold, at least about 200 fold, or as much asnearly about 300 fold, about 400 fold or about 500 fold, during thecourse of treatment. For example, IL5RA mRNA levels in AML HTCs isalmost 750 times that in normal HSCs (see Table 1). A reduction to 200,100, 50, 20, 10, or just 2 times the IL5RA mRNA levels in normal HSCs isevidence of an effective therapeutic regime.

In some embodiments, the methods of the present invention are suitablefor diagnosis prognosis and/or monitoring of a haematologicalproliferative disorder of myeloid origin, such as myoproliferativedisorders. The present invention also provides methods of diagnosis,prognosis and monitoring of a myoproliferative disorder that is chronicmyeloid leukemia (CML) and/or acute myeloid leukemia (AML). In aparticularly preferred embodiment, the myelogenous haematologicalproliferative disorder is AML and the level of RNA or antigen isdetected using a test sample comprising AML HTCs, compared to controllevels obtained from a control sample of normal HSCs.

9. KITS

In another aspect of the present invention, kits are provided whichcontain one or more of the necessary reagents to carry out methods ofthe present invention. Specifically, some embodiments provide acompartment kit having one or more containers, which comprise: (a) afirst container comprising one of the marker specific antibodies orcomplexes of the present invention, e.g., a first monoclonal antibodythat specifically binds a first antigen corresponding to one of thecytokine receptors disclosed herein; and (b) one or more othercontainers comprising one or more of the following: wash reagents,reagents capable of detecting presence of the antibody, and/or anothermarker specific antibody or complex of the present invention, e.g., asecond monoclonal antibody that specifically binds a second antigencorresponding to a different cytokine receptor disclosed herein. In someembodiments, kits include additional containers, e.g, comprising third,fourth, fifth, etc., antibodies directed to antigens corresponding to athird, fourth, fifth, etc., cytokine receptor disclosed herein. In someembodiments, additional containers include one or more known smallmolecule agonists or antagonists that find use in the prognostic,diagnostic and/or therapeutic methods taught herein.

The kit typically contains containers which may be formed from a varietyof materials such as glass or plastic, and can include, for example,bottles, vials, syringes, and test tubes. A compartment kit includes anykit in which reagents are contained in separate containers. Suchcontainers include small glass containers, plastic containers or stripsof plastic or paper. Such containers allow efficient transfer ofreagents from one compartment to another compartment, such that thesamples and reagents are not cross-contaminated, and the agents orsolutions of each container can be added in a quantitative fashion fromone compartment to another. One skilled in the art will readilyrecognize that the disclosed antibodies of the present invention can bereadily incorporated into one of the established kit formats, which arewell known in the art.

Provided herein are kits which include a composition described herein.In some embodiments the kit comprises a hybridoma, complex, antibodyand/or mixtures of antibodies disclosed herein. In some embodiments,kits for therapeutic applications are provided, such as a kit housing apharmaceutical formulation, e.g., one or more of the pharmaceuticalcompositions described herein. In some embodiments, the kits contain atleast one additional reagent, including other antibodies, othermonoclonal antibodies directed to HSCs, other agents described herein,committed progenitor cells, polyclonal antibodies, or mixtures of theantibodies as reagents for detecting myeloid cell types. Frozen or fixedforms of HSCs, CMPs, GMP and/or MEPs reactive with the antibodies andreagents form additional contents of the kits.

In some embodiments, the kit is a diagnostic kit for use in detectingtest samples. The kit can include a control antibody that does not reactwith the antigen to be assayed, along with a marker specific antibody orantigen-binding fragment thereof which specifically binds to an antigencorresponding to a cytokine receptor disclosed herein. Further, such akit can include a means for detecting the binding of said antibody tothe antigen (for example, the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry).

In preferred embodiments, the diagnostic kit includes a substantiallyisolated antibody that specifically binds an antigen corresponding to anHTC marker (e.g., a cytokine receptor disclosed herein), as well asmeans for detecting antigen-antibody binding. In some embodiments, theantibody is attached to a solid support. In some embodiments, theantibody is a monoclonal antibody. The detecting means of the kit caninclude a second, labeled monoclonal antibody. Alternatively, or inaddition, the detecting means can include a labeled, competing antigen.

In one diagnostic configuration, the test sample is reacted with a solidphase reagent having a surface-bound antigen, where the antigencorresponds to one (or more) of the cytokine receptors disclosed herein.After washing to removing unbound components, the reagent can be reactedwith reporter-labeled anti-human antibody to determine the amount ofanti-antigen antibody bound to the solid support. Such methods are wellknown and have been extensively described in the art. The reporter labelcan be an enzyme, for example, which is detected by incubating the solidphase with a suitable fluorometric, luminescent or calorimetricsubstrate, as is standard in the art.

The solid surface bearing bound antigens and/or antibodies, as describedabove, can be prepared by known techniques for attaching proteinmaterial to solid support material. Suitable solid support materialsinclude, for example and without limitation, polymeric beads, dipsticks, 96-well plate or filter material. In some embodiments, a smallmolecule known to bind to a polypeptide corresponding to a disclosedcytokine receptor can be attached to a solid support, based on itschemical structure by methods known in the art.

A text label typically accompanies the kit, and includes any writing orrecorded material, which may be electronic or computer readable form(e.g., disk, optical disc, or tape) providing instructions or otherinformation for using one or more of the contents of the kit. In someembodiments, the label indicates that the contents are used fordiagnosing or treating the disorder of choice, such as a hematopoieticproliferatvie disorder of myeloid origin, according to one or more ofthe methods described herein. In some embodiments, AML represents thedisorder to be diagnosed and/or treated using the contents of the kit.In some embodiments, CML represents the disorder to be diagnosed and/ortreated using the contents of the kit.

10. EXAMPLES 10.1 Example 1 Identification of Cytokine ReceptorsAssociated with HTCs

HTC markers were identified by comparing RNA transcript levels in normalHSCs and in AML CSCs for a variety of genes using micorarrays.Specifically, data was obtained for test samples comprising AMLLin⁻CD34⁺CD38⁻ cells, where three AML samples were taken from peripheralblood; Lin⁻CD34⁺CD38⁻ and Lin⁻CD34⁺CD38⁺ cells were double sorted. Thesorting strategy produced cells which were also over 90% CD90⁻. Thesorting strategy produced purities greater than 98%.

To allow for direct comparison, data was then obtained from controlsamples comprising normal HSCs. A sample was taken from mobilizedperipheral blood (MPB) of each of three individual donors andLin⁻CD34⁺CD90⁺CD45RA⁻CD38⁻ and Lin⁻CD34⁺CD90⁺CD45RA⁻CD38⁺ cells weredouble sorted. The sorting strategy produced a purity of over 99%.

For both sorting strategies, the Lineage (Lin) cocktail includedantibodies against CD2, CD3, CD11b, CD15, CD19, CD41, and CD235.Exemplary dot plots of control samples and AML samples are shown inFIGS. 1 and 2, respectively.

Total RNA was then extracted, the RNA was reverse transcribed and invitro transcribed to ultimately yield fluorochrome labeled cRNA probesfrom the transcripts. Transcript levels were detected using Affymetrixwhole Human Genome U133 Plus 2.0 Array. Briefly, the gene array washybridized and read out. Signal intensities, probe set ID # andabsence/presence score for each probe set was tabulated using MS Excel.

The signal values corresponding to RNA transcript levels in AML testsamples and control samples (Lin⁻CD34⁺CD90⁺CD45RA⁻CD38⁻ normal HSCs)were then directly compared using MS Excel and Fisher's t-test.

HTC marker genes were then selected based on the following criteria: (1)significance of signal intensity difference between test and controlsamples cohorts p<0.05; (2) genes not expressed in all 3 normal HSCsamples (scored ‘absent’ by Affymetrix software); (3) genes expressed inat least 2 out of 3 AML samples (scored ‘present’ by Affymetrixsoftware); (4) AMUHSC signal ratio ≧5 or 5) genes whose expression wasknown to be extracellular and a cytokine receptor.

Tables 1 and 2 below shows comparison for 10 genes that wereover-expressed in at least 2 out of the 3 of the CD38⁻ or CD38⁺,respectively, AML CSC samples compared to all three of the normal HSCsamples. “Pos. samples” refers to positive samples, scored as ‘present’by Affymetrix software (in the case of normal HSCs) or giving a signalintensity greater than 25 (in the case of AML CSCs).

TABLE 1 Normal CD38⁻ CD38⁻ AML HSC sample CSC sample Pos. Average Pos.Average samples signal samples signal Signal Gene detected intensitydetected intensity ratio CSF1R 0/3 26 2/3 89 3 IFNAR1 0/3 30 2/3 59 2IL13RA1 0/3 14 3/3 467 33 IL1RAP 0/3 25 3/3 411 16 IL5RA 0/3 8 2/3 6000750 INSR 0/3 49 3/3 132 3 IL1RL1 0/3 18 2/3 342 19 LTK 0/3 39 2/3 126 3TACSTD1 3/3 189 2/3 1561 8

TABLE 2 Normal CD38⁺ CD38⁺ AML HSC sample CSC sample Pos. Average Pos.Average samples signal samples signal Signal Gene detected intensitydetected intensity ratio CSF1R 0/3 36 2/3 193 5 IFNAR1 0/3 23 3/3 112 5IL13RA1 3/3 179 3/3 606 3 IL1RAP 1/3 54 3/3 803 15 IL5RA 0/3 7 2/3 1137162 LILRA1 0/3 13 2/3 44 3 INSR 0/3 33 3/3 192 6 IL1RL1 0/3 50 3/3 136 7LTK 0/3 28 2/3 132 5 TACSTD1 3/3 201 2/3 2474 12

Comparison of published data by Gal et al. (“Gene expression profiles ofAML derived stem cells; similarity to hematopoietic stem cells.”Leukemia 2006, 20: 2147-2154, incorporated herein by reference in itsentirety) with control samples described herein corroborates use ofCFS1R, and LTK as cancer stem cell targets (See U.S. Patent ApplicationNo. 61/039,701, incorporated herein by reference).

As shown in FIG. 3, f expression of IL1RAP by AML CSCs was detectably byflow cytometric analysis using the 3D8 antibody from Novus Biological(Littleton, Colo.). In contrast, expression of IL1RAP by normal HSCs isundetectable by flow cytometric analysis (FIG. 3).

10.2 Example 2 Production and In Vivo Efficacy of Monoclonal Antibodies10.2.1. Preparation of Monoclonal Antibodies that Specifically Bind anCytokine Receptor Associated with HTCs

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986). Immunogensthat may be employed include a purified polypeptide corresponding tocolony stimulating factor 1 receptor (CFS1R); interleukin 13 receptor,alpha 1 (IL13RA1); interleukin 1 receptor accessory protein (IL1RAP);interferon-α receptor 1 (IFNAR1); interleukin-5 receptor (IL5R); insulinreceptor (INSR); interleukin 1 receptor-like 1 (IL1RL1);leukocytereceptor tyrosine (LTK); or tumor associated calcium signal transducer 1(TACSTD1) as well as fusion proteins containing the same. Alternatively,cells expressing recombinant CFS1R; IL13RA1; IL1RAP; IFNAR1, IL5RA,INSR, IL1RL1, LTK or TACSTD1, on the cell surface, may be used.Selection of the immunogen can be made by the skilled artisan withoutundue experimentation.

Mice, such as Balb/c, are immunized with the selected immunogen,emulsified in complete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectantibodies directed to the HTC marker polypeptide.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of the immunogen corresponding to an HTC marker. Three to fourdays later, the mice are sacrificed and the spleen cells harvested. Thespleen cells are then fused (using 35% polyethylene glycol) to aselected murine myeloma cell line such as P3X63AgU.1, available fromATCC, No. CRL 1597. The fusions generate hybridoma cells which can thenbe plated in 96 well tissue culture plates containing HAT (hypoxanthine,amininopterin, and thymidine) medium to inhibit proliferation ofnon-fused cells, myeloma hybrids, and spleen cell hybrids.

The hybridoma cells then can be screened in an ELISA for reactivityagainst the immunogen corresponding to the HTC marker. Determination of“positive” hybridoma cells secreting the desired monoclonal antibodiesdirected against the HTC marker polypeptide is within the skill in theart.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the monoclonalantibodies directed to polypeptide corresponding to the HTC marker.Alternatively, the hybridoma cells can be grown in tissue culture flasksor roller bottles. Purification of the monoclonal antibodies produced inthe ascites can be accomplished using ammonium sulfate precipitation,followed by gel exclusion chromatography, as known in the art.Alternatively, affinity chromatography based upon binding of antibody toprotein A or protein G can be employed, also as known in the art.

10.2.2. In Vivo Efficacy of Monoclonal Antibodies Against MyeloidLeukemia

In vivo models of human cancer are useful to determine preclinicalefficacy of candidate therapeutic agents. For monoclonal antibodies,studies in appropriate animal models help evaluate target cell lysis andtumor eradication under physiological conditions in vivo. Several groupshave described engraftment of CML chronic phase (CP), accelerated phase(AP), and/or blast phase (BP) and AML cells into SCID and NOD/SCID mice.In general, generation of chimeric animals showing engraftment of humanCML cells is more consistent in NOD/SCID mice (See Dazzi, F et al, Blood92: 1390-1396 (1998); Wang, J. C. Y. et al, Blood 91: 2406-2414 (1998);Dick, J et al Blood 87: 1539-1548 (1996); Bonnet, D et al, Blood 106:4086-4092 (2005)). In vivo efficacy of monoclonal antibodies against CMLand/or normal GMP and not HSC can be determined using the NOD/SCID humanCML model.

Xenograft animals can be generated as described by Dazzi et al. Briefly,NOD/SCID mice are bred in house or purchased from a commercial supplier(Jackson Laboratories) and housed under pathogen-free conditions. Priorto injection of cells, animals are irradiated (250 cGy, x-ray source).Cryopreserved cells from a CML or AML patient obtained from peripheralblood, mobilized peripheral blood, or bone marrow are analyzed by flowcytometry to determine the percentage of CD34⁺ cells in the sample.Samples containing 1 to 10×10⁶ CD34⁺ cells are injected IV into theconditioned mouse in a total volume of 1 mL. Alternatively, CD34⁺ cellscan be sorted from the sample by FACS prior to transplantation. A subsetof the animals is sacrificed weekly and bone marrow and spleen analyzedfor human CD34⁺ cells.

Patient samples with engraftment potential are selected for use inantibody efficacy studies. For efficacy studies, CMUAML cells aretransplanted and the test monoclonal antibody or control antibody willbe injected on a schedule. Alternate schedules include once to 3 timesper week, 1-3 injections per week for 1-4 weeks, or 1-2 per week for 1-4months. Injections can be intravenous by tail vein injection,intraperitoneal, subcutaneous, or intramuscular. Following completion ofthe treatment schedule, animals are sacrificed and tissues collected foranalysis. Peripheral blood, spleen and bone marrow can be evaluated byFACS analysis for the presence of human phenotypic CML cancer stemcells, CD34⁺ marker⁺ HTCs, detectable in the bone marrow and spleen atthe conclusion of the treatment. Philadelphia (Ph) chromosome can beassayed by PCR to determine whether the cells are CML or normal.

As an example, eleven mice can be transplanted with CML sample (MISIRB31104 750), 5×10⁶ cells/mouse. Mice can be conditioned with 250 rad TBI(x-ray source, Faxitron CP160), and anti-asialo GM1. The anti-asialo GM1is injected by intraperitoneal injection on days 0, 5 and 11. At 4 weekspost transplant half the mice in each group will begin receivingintraperitoneal or intravenous injections of a clone of a markerspecific mAb, described herein, or control antibody, 250-1000 mg/dose,two times a week for 4 weeks. Additionally, a group of 40 mice are alsotransplanted with CML (MISIRB 31104 750) as above. Antibodyadministration begins at the time of transplant. Mice are injected byintraperitoneal injection with 0.5-1 mg/dose of antibody, twice a weekfor 8 weeks. Alternatively, CML cells isolated from previously engraftedmice will be serially transplanted. Some of these secondary recipientswill be treated with the marker specific mAb clone at time oftransplantation by intraperitoneal injection with 0.5-1 mg/dose ofantibody, twice a week for 8 weeks. Following the above treatments withthe marker specific mAb clone, the mice are analyzed by flow cytometryfor tumor burden, expression of CD34 and expression of the specific HTCmarker gene. The number and frequency of human cells in the bone marrowand spleen will be determined for all mice surviving to the end of thestudy. Human cells (marker+) will be sorted by FACS from both groups ofmice for serial transplantation to determine if cells with functionalcancer stem cell potential are present.

For secondary transplant of CML cells, CD34⁺marker⁺ cells can be sortedfrom the bone marrow and spleen of several mice for transplantation.NOD/SCID analysis for the secondary transplant can be performed 8 or 10weeks post transplant with the CD34 compartment of bone marrow.

Determination of whether the marker specific monoclonal antibody clonecan eliminate or reduce tumor burden in mice transplanted with primaryhuman AML blast crisis cells. Twenty-five mice can be transplanted withAML cells injected at 10×10⁶ CD34⁺marker⁺ cells/mouse. Cells areinjected intravenously into the tail vein or the post lateral aspect ofthe orbital cavity. Mice are conditioned with 250 rad TBI (x-ray source,Faxitron CP160), and anti-asialo GM1. The anti-asialo GM1 is injected IPon days 0, 5 and 11. Beginning 4 weeks post transplant, mice arerandomized into 2 groups, therapy and control. The group receiving thetherapeutic is injected I.P. with the marker specific mAb clone.Antibody will be administered by intraperitoneal injection 2 times aweek, for 4 weeks. Antibody concentration will be 1 mg per injection (atotal of 2 mg/mouse/week). Volume will vary depending on the antibodylot used. Mouse IgG will be used as the control article; it will beprepared in the same diluent and injected at the same concentration andvolume as the monoclonal antibody. Mice will be sacrificed 2-3 daysfollowing the last injection of marker specific antibody clone. Bonemarrow and spleen will be isolated and counted. Tissues will be analyzedby FACS for expression of CD34 and the specific HTC marker gene. Thenumber and frequency of human cells in the bone marrow and spleen willbe determined for all mice surviving to the end of the study. Humancells will be sorted by FACS from both groups of mice for serialtransplantation to determine if cells with functional cancer stem cellpotential are present post-antibody treatment. Alternatively, mice willbegin antibody treatment at the time of AML cell transplant. These micewill be injected with 0.5mg of antibody 2 times a week for 8 weeks.Analysis will proceed as described above.

In addition, the efficacy of the maker specific mAb clone can be testedfor efficacy in leukemia in vivo models using human cell linesexpressing the corresponding antigen. Immunocompromised animals will beinoculated with human leukemia cell lines recognized by the markerspecific mAb clone. The efficacy of the clone will be tested usingmultiple cell lines. The cell lines used should maintain a primitivephenotype in vivo with sustained expression of the epitope recognized bythe marker specific mAb clone. Such cell lines will be identified andused as appropriate. Each cell line will be characterized usingdifferent routes of administration; intravenous, subcutaneous orintraperitoneal injection. NOD/SCID mice will be tested for tumorengraftment at the site of injection and subsequent invasion of bonemarrow and spleen. Animals will be treated with the marker specific mAbclone beginning at the time of tumor inoculation or following tumorengraftment with injections starting 1-4 weeks post cell administration.Antibody will be administered by intraperitoneal injection 2 times aweek, for 4 weeks. Mouse IgG will be used as the control article,injected at the same concentration and volume as the marker specific mAbclone. Test cells will be administered at a single site. Animals areweighed weekly and observed for clinical signs of toxicity and death forthe duration of treatment. Animals are observed and palpated for theformation of nodules at the site of injection twice weekly. Detectednodules will be measured in two dimensions and findings recorded. Theinjection site is exposed at the end of study and tumor removed andmeasured and weighed. In addition, a sample of bone marrow, spleen andtumor mass are to be removed for phenotyping by FACS. These tissues willbe disassociated and prepared for analysis by flow cytometry. Tissueswill be screened for expression of one or more of the human HTC markersand the marker specific mAb clone.

Using the above described in vivo models, it can be shown that treatmentwith monoclonal antibody compositions of the present invention iseffective in reducing tumor size, ameliorating one or more symptomsand/or prolonging survival of mice in the therapy group.

The foregoing descriptions of specific embodiments of the presentinvention have been presented for purposes of illustration anddescription. They are not intended to be exhaustive or to limit theinvention to the precise forms disclosed, and obviously manymodifications and variations are possible in light of the aboveteaching. The embodiments were chosen and described in order to bestexplain the principles of the invention and its practical application,to thereby enable others skilled in the art to best utilize theinvention and various embodiments with various modifications as aresuited to the particular use contemplated. It is intended that the scopeof the invention be defined by the Claims appended hereto and theirequivalents.

All patents, patent applications, publications, and references citedherein are expressly incorporated by reference to the same extent as ifeach individual publication or patent application was specifically andindividually indicated to be incorporated by reference.

What is claimed is:
 1. A method of monitoring the efficacy of treatingacute myelogenous leukemia (AML) in a patient, comprising obtaining atest sample from said patient at two or more time points during saidtreatment of AML, wherein the test sample is a blood sample detecting alevel of an expression product corresponding to interleukin 1 receptoraccessory protein (IL1RAP) in CD34+ cells in each of the test samples,and determining that the patient has a positive response to treatment ofAML when the level in the levels of said expression product are reducedover time.
 2. The method of claim 1, wherein said expression product ismRNA.
 3. The method of claim 1, wherein the levels of at least twodifferent expression products are detected at two or more of said timepoints.
 4. The method of claim 1, wherein said detecting uses amicroarray.
 5. The method of claim 1, wherein the expression product isprotein.
 6. The method of claim 5, wherein the detecting comprises useof an antibody.